Anti-CCR2 antibodies and methods of use therefor

ABSTRACT

The present invention relates to an antibody or functional fragment thereof which binds to a mammalian (e.g., human) CC-chemokine receptor 2 (CCR2) or a portion of the receptor and blocks binding of a ligand to the receptor. The invention further relates to a method of inhibiting the interaction of a cell bearing mammalian CCR2 with a ligand thereof, and to use of the antibodies and fragments in therapeutic, prophylactic and diagnostic methods.

RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 09/121,781, filed Jul. 23, 1998, the entire teachings of whichare incorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION

Over the past several years a growing family of leukocytechemoattractant/activating factors, termed chemokines, has beendescribed (Oppenheim, J. J. et al., Annu. Rev. Immunol., 9:617-648(1991); Schall and Bacon, Curr. Opin. Immunol., 6:865-873 (1994);Baggiolini, M., et al., Adv. Imunol., 55:97-179 (1994)). Members of thisfamily are produced and secreted by many cell types in response to earlyinflammatory mediators such as IL-1β or TNFα. The chemokine superfamilycomprises two main branches: the α-chemokines (or CXC chemokines) andthe β-chemokines (CC chemokines). The α-chemokine branch includesproteins such as IL-8, neutrophil activating peptide-2 (NAP-2), melanomagrowth stimulatory activity (MGSA/gro or GROα), and ENA-78, each ofwhich have attracting and activating effects predominantly onneutrophils. The members of the β-chemokine branch affect other celltypes such as monocytes, lymphocytes, basophils, and eosinophils(Oppenheim, J. J. et al., Annu. Rev. Immunol., 9:617-648 (1991);Baggiolini, M., et al., Adv. Imunol., 55:97-179 (1994); Miller andKrangel, Crit. Rev. Immunol., 12:17-46 (1992); Jose, P. J., et al., J.Exp. Med., 179:881-118 (1994); Ponath, P. D., et al., J. Clin. Invest.,97:604-612 (1996)), and include proteins such as monocyte chemotacticproteins 1-4 (MCP-1, MCP-2, MCP-3, and MCP-4), RANTES, and macrophageinflammatory proteins (MIP-1α, MIP-1β). Recently a new class ofmembrane-bound chemokines designated CX3C chemokines has been identified(Bazan, J. F., et al., Nature 385:640-644 (1997)). Chemokines canmediate a range of pro-inflammatory effects on leukocytes, such astriggering of chemotaxis, degranulation, synthesis of lipid mediators,and integrin activation (Oppenheim, J. J. et al., Annu. Rev. Immunol.,9:617-648 (1991); Baggiolini, M., et al., Adv. Imunol., 55:97-179(1994); Miller, M. D. and Krangel, M. S., Crit. Rev. Immunol., 12:17-46(1992)). Lately, certain β-chemokines have been shown to suppress HIV-1infection of human T cell lines in vitro (Cocchi, F., et al., Science(Wash. D.C.), 270:1811-1815 (1995)).

Chemokines bind to 7 transmembrane spanning (7TMS) G protein-coupledreceptors (Murphy, P. M., Annu. Rev. Immunol., 12:593-633 (1994)). Someknown receptors for the CC or β chemokines include CCR1, which bindsMIP-1α and RANTES (Neote, K., et al., Cell, 72:415-425 (1993); Gao, J.L., J. Exp. Med., 177:1421-1427 (1993)); CCR2, which binds chemokinesincluding MCP-1, MCP-2, MCP-3 and MCP-4 (Charo, I. F., et al., Proc.Natl. Acad. Sci. USA, 91:2752-2756 (1994); Myers, S. J., et al., J.Biol. Chem., 270:5786-5792 (1995); Gong et al., J. Biol Chem272:11682-11685 (1997); Garcia-Zepeda et al., J. Immunol. 157:5613-5626(1996)); CCR3, which binds chemokines including eotaxin, RANTES andMCP-3 (Ponath, P. D., et al., J. Exp. Med., 183:2437-2448 (1996)); CCR4,which has been found to signal in response to MCP-1, MIP-1α, and RANTES(Power, C. A., et al., J. Biol. Chem., 270:19495-19500 (1995)); andCCR5, which has been shown to signal in response to MIP-1α, MIP-1β andRANTES (Boring, L., et al., J. Biol. Chem., 271 (13):7551-7558 (1996);Raport, C. J., J. Biol. Chem., 271:17161-17166 (1996); and Samson, M. etal., Biochemistry, 35:3362-3367 (1996)).

CCR2 is expressed on the surface of several leukocyte subsets, andappears to be expressed in two slightly different forms (CCR2a andCCR2b) due to alternative splicing of the mRNA encoding thecarboxy-terminal region (Charo et al., Proc. Natl. Acad. Sci. USA91:2752-2756 (1994)). MCP-1 acts upon monocytes, lymphocytes andbasophils, inducing chemotaxis, granule release, respiratory burst andhistamine and cytokine release. Studies have suggested that MCP-1 isimplicated in the pathology of diseases such as rheumatoid arthritis,atherosclerosis, granulomatous diseases and multiple sclerosis (Koch, J.Clin. Invest. 90:772-79 (1992); Hosaka et al., Clin. Exp. Immunol.97:451-457 (1994); Schwartz et al., Am. J. Cardiol. 71(6):9B-14B (1993);Schimmer et al., J. Immunol. 160:1466-1471 (1998); Flory et al., Lab.Invest. 69:396-404 (1993); Gong et al., J. Exp. Med. 186:131-137(1997)). Additionally, CCR2 can act as a co-receptor for HIV (Connor etal., J. Exp. Med. 185:621-628 (1997)). Thus, CCR2 receptor antagonistsmay represent a new class of important therapeutic agents.

SUMMARY OF THE INVENTION

The present invention relates to an antibody (immunoglobulin) orfunctional fragment thereof (e.g., an antigen-binding fragment) whichbinds to a mammalian CC-chemokine receptor 2 (also referred to as CCR2,CKR-2, MCP-1RA or MCP-1RB) or portion of the receptor (anti-CCR2). Inone embodiment, the antibody of the present invention or fragmentthereof has specificity for human or rhesus CCR2 or a portion thereof.In another embodiment, the antibody or fragment of the invention blocksbinding of a ligand (e.g., MCP-1, MCP-2, MCP-3, MCP-4) to the receptorand inhibits function associated with binding of the ligand to thereceptor (e.g., leukocyte trafficking). For example, as describedherein, antibodies and fragments thereof of the present invention whichbind human or rhesus CCR2 or a portion thereof, can block binding of achemokine (e.g., MCP-1, MCP-2, MCP-3, MCP-4) to the receptor and inhibitfunction associated with binding of the chemokine to the receptor. Inone embodiment, the antibody is monoclonal antibody (mAb) LS132.1D9(1D9) or an antibody which can compete with 1D9 for binding to humanCCR2 or a portion of human CCR2. Functional fragments of the foregoingantibodies are also envisioned.

In another embodiment, the antibody or functional fragment of thepresent invention binds human CCR2 or a portion thereof, and inhibitshuman immunodeficiency virus (HIV) binding to the receptor, therebyinhibiting function associated with binding of HIV to the receptor(e.g., HIV antigen release and infectivity). In one embodiment, theantibody is monoclonal antibody 1D9 or an antibody which can competewith 1D9 for binding to human CCR2 or a portion of human CCR2.

The present invention also relates to an antibody or functional fragmentthereof (e.g., an antigen-binding fragment) which binds to a mammalianCCR2 or portion of the receptor and provides increased fluorescentstaining intensity of CCR2 or compositions comprising CCR2 relative toother anti-CCR2 antibodies. In one embodiment, the antibody ismonoclonal antibody 1D9 or LS132.8G2 (8G2) or an antibody which cancompete with 1D9 or 8G2 for binding to human CCR2 or a portion of humanCCR2.

The present invention further relates to a method of inhibiting theinteraction of a cell bearing mammalian (e.g., human, non-human primateor murine) CCR2 with a ligand thereof, comprising contacting the cellwith an effective amount of an antibody or functional fragment thereofwhich binds to a mammalian CCR2 or a portion of CCR2. Suitable cellsinclude granulocytes, leukocytes, such as monocytes, macrophages,basophils and eosinophils, mast cells, and lymphocytes including T cells(e.g., CD8+ cells, CD4+ cells, CD25+ cells, CD45RO+ cells), and othercells expressing CCR2 such as a recombinant cell expressing CCR2 (e.g.,transfected cells). In a particular embodiment, the antibody is 1D9 oran antibody which can compete with 1D9 for binding to human CCR2 or aportion of human CCR2.

Another embodiment of the invention relates to a method of inhibitingthe interaction of a cell bearing mammalian CCR2 with a chemokine,comprising contacting said cell with an effective amount of an antibodyor functional fragment thereof which binds to CCR2 or a portion of saidreceptor. In one embodiment of the method, the antibody or functionalfragment thereof is any one or more of 1D9, an antigen-binding fragmentof 1D9 or an antibody or fragment thereof having an epitopic specificitywhich is the same as or similar to that of 1D9. Furthermore, theinvention relates to a method of inhibiting a function associated withbinding of a chemokine to CCR2, comprising administering an effectiveamount of an antibody or functional fragment thereof which binds to amammalian CCR2 protein or a portion of said receptor. In one aspect ofthe method, the antibody or functional fragment thereof is any one ormore of 1D9, an antigen-binding fragment of 1D9 or an antibody orfragment thereof having an epitopic specificity which is the same as orsimilar to that of 1D9.

Another aspect of the invention is a method of identifying expression ofa mammalian CCR2 or portion of the receptor by a cell. According to themethod, a composition comprising a cell or fraction thereof (e.g., amembrane fraction) is contacted with an antibody or functional fragmentthereof (e.g., 1D9 or 8G2) which binds to a mammalian CCR2 protein orportion of the receptor under conditions appropriate for binding of theantibody thereto, and the formation of a complex between said antibodyor fragment and said protein or portion thereof is detected. Detectionof the complex, directly or indirectly, indicates the presence of thereceptor on the cell. The present invention also relates to a kit foruse in detecting the presence of CCR2 or a portion thereof in abiological sample, comprising an antibody or functional fragment thereofwhich binds to a mammalian CC-chemokine receptor 2 or a portion of saidreceptor, and one or more ancillary reagents suitable for detecting thepresence of a complex between said antibody or fragment and said proteinor portion thereof.

Also encompassed by the present invention are methods of identifyingadditional ligands or other substances which bind a mammalian CCR2protein, including inhibitors and/or promoters of mammalian CCR2function. For example, agents having the same or a similar bindingspecificity as that of an antibody of the present invention orfunctional fragment thereof can be identified by a competition assaywith said antibody or fragment. Thus, the present invention alsoencompasses methods of identifying ligands or other substances whichbind the CCR2 receptor, including inhibitors (e.g., antagonists) orpromoters (e.g., agonists) of receptor function. In one embodiment,cells which naturally express CCR2 receptor protein or suitable hostcells which have been engineered to express a CCR2 receptor or variantencoded by a nucleic acid introduced into said cells are used in anassay to identify and assess the efficacy of ligands, inhibitors orpromoters of receptor function. Such cells are also useful in assessingthe function of the expressed receptor protein or polypeptide.

Thus, the invention also relates to a method of detecting or identifyingan agent which binds a mammalian CCR2 or ligand binding variant thereof,comprising combining an agent to be tested, an antibody orantigen-binding fragment of the present invention (e.g., monoclonalantibody 1D9, an antibody having an epitopic specificity which is thesame as or similar to that of 1D9, antigen-binding fragments of 1D9,monoclonal antibody 8G2, an antibody having an epitopic specificitywhich is the same as or similar to that of 8G2, and antigen-bindingfragments of 8G2) and a composition comprising a mammalian CCR2 proteinor a ligand binding variant thereof. The foregoing components can becombined under conditions suitable for binding of the antibody orantigen-binding fragment to mammalian CCR2 protein or a ligand bindingvariant thereof, and binding of the antibody or fragment to themammalian CCR2 protein or ligand binding variant is detected ormeasured, either directly or indirectly, according to methods describedherein or other suitable methods. A decrease in the amount of complexformed relative to a suitable control (e.g., in the absence of the agentto be tested) is indicative that the agent binds said receptor orvariant. The composition comprising a mammalian CCR2 protein or a ligandbinding variant thereof can be a membrane fraction of a cell bearingrecombinant CCR2 protein or ligand binding variant thereof. The antibodyor fragment thereof can be labeled with a label such as a radioisotope,spin label, antigen label, enzyme label, fluorescent group andchemiluminescent group. These and similar assays can be used to detectagents, including ligands (e.g., chemokines which interact with CCR2) orother substances, including inhibitors or promoters of receptorfunction, which can bind CCR2 and compete with the antibodies describedherein for binding to the receptor.

According to the present invention, ligands, inhibitors or promoters ofreceptor function can be identified in a suitable assay, and furtherassessed for therapeutic effect. Inhibitors of receptor function can beused to inhibit (reduce or prevent) receptor activity, and ligandsand/or promoters can be used to induce (trigger or enhance) normalreceptor function where indicated. The present invention also provides amethod of treating inflammatory diseases, autoimmune diseases,atherosclerosis, and graft rejection, or HIV infection, comprisingadministering an inhibitor of receptor function (e.g., chemokine bindingor HIV binding) to an individual (e.g., a mammal, such as a human). Thepresent invention further provides a method of stimulating receptorfunction by administering a novel ligand or promoter to an individual,providing a new approach to selective stimulation of leukocyte function,which is useful, for example, in the treatment of infectious diseasesand cancer.

Another aspect of the invention relates to a method of inhibiting HIVinfection of a cell which expresses a mammalian CCR2 or portion thereof,comprising contacting the cell with an effective amount of an antibodyor functional fragment thereof which binds to a mammalian CCR2 orportion of the receptor and inhibits HIV binding and infection. In aparticular embodiment of the invention, the antibody or functionalfragment thereof is any of 1D9, an antibody having an epitopicspecificity which is the same as or similar to that of 1D9, an antibodywhich can compete with 1D9 for binding to human CCR2, andantigen-binding fragments thereof.

Also encompassed by the present invention is a method of inhibiting(e.g., treating) HIV in a patient, comprising administering to thepatient an effective amount of an antibody or functional fragmentthereof which binds to a mammalian CCR2 or a portion of said receptorand inhibits HIV binding to the CCR2 receptor. The anti-CCR2 antibody orfragment can be administered alone or in combination with one or moreadditional therapeutic agents, e.g., one or more antibodies which bind aco-receptor for HIV infection and inhibit binding to said co-receptor,such as an anti-CCR3, anti-CCR5, and/or anti-CXCR4 antibody.

Another aspect of the invention also relates to a method of preventingor inhibiting HIV infection in an individual, comprising administeringto the individual an effective amount of an antibody or functionalfragment thereof which binds to CCR2 and inhibits HIV binding to CCR2.According to the method, preventing HIV infection includes treatment inorder to prevent (reduce or eliminate) infection of new cells in aninfected individual or in order to prevent infection in an individualwho may be, may have been or has been exposed to HIV. For example,individuals such as an HIV infected individual, a fetus of an HIVinfected female, or a health care worker can be treated according to themethod of the present invention.

The present invention also encompasses a method of inhibiting leukocytetrafficking in a patient, comprising administering to the patient aneffective amount of an antibody or functional fragment thereof whichbinds to a mammalian CCR2 or portion of said receptor and inhibitsfunction associated with binding of a ligand to the receptor.

The present invention also relates to a method of inhibiting or treatingCCR2-mediated disorders, such as inflammatory disorders, comprisingadministering to a patient an effective amount of an antibody orfunctional fragment thereof which binds to a mammalian CCR2 or portionof said receptor and inhibits CCR2-mediated function. For example, theinvention relates to a method of inhibiting or treating stenosis orrestenosis of the vasculature comprising administering to a patient aneffective amount of an antibody or functional fragment thereof whichbinds to a mammalian CCR2 or portion of said receptor and inhibitsCCR2-mediated function.

The present invention further relates to an antibody or fragment thereofas described herein (e.g., monoclonal antibody 1D9 or an antigen-bindingfragment thereof) for use in therapy (including prophylaxis) ordiagnosis, and to the use of such an antibody or fragment for themanufacture of a medicament for the treatment of a CCR2-mediateddisorder, or other disease or inflammatory condition as describedherein.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1O fluorescence activated cell scanning (FACS) histogramprofiles illustrating that mAbs 1D9 and 8G2 stain CCR2 transfectants butnot CCR5 or CCR1 transfectants. L1/2 (also referred to herein as L1.2)murine pre-B lymphoma host cells were transfected with CCR2, CCR5 andCCR1 as indicated, and stained with antibodies with different receptorspecificities. Staining was analyzed by flow cytometry.

FIGS. 2A-2L FACS dot plots showing expression of CCR2 on most monocytes,a subpopulation of lymphocytes and a small subset of granulocytes. Wholeblood cells were stained with one of three anti-CCR2 mAbs (5A11,generated using a peptide consisting of the first 32 amino acids of theCCR2 amino-terminus as an immunogen, and 1D9 and 8G2 generated asdescribed herein using CCR2b L1/2 cell transfectants as the immunogen).Staining was analyzed by flow cytometry, and the lymphocyte, granulocyteand monocyte populations were gated using the forward and side lightscatter. The X-axis represents forward light scatter (a measure of cellsize), and the Y-axis fluorescence intensity of staining for CCR2. Thelevel of negative control staining is indicated by a line.

FIGS. 3A-3I FACS dot plots showing that mAb 1D9 stains an IgE positivepopulation in peripheral blood (basophils) using two-color staining forIgE and CCR2. Whole blood cells were first stained with either anegative control antibody (anti-Flag), anti-CCR2 antibody 1D9, or ananti-CXCR1 antibody, as indicated, and detected by an anti-mouse-FITCconjugate. A second staining was done using either PBS or a biotinylatedantibody specific for IgE or CD16, as indicated, and detected with astreptavidin-phycoerythrin. Staining was analyzed by flow cytometry.

FIG. 4 illustrates that mAb 1D9 inhibits [¹²⁵I]MCP-1 binding to THP-1cell membranes. 3.0 μg of THP-1 membrane protein was incubated with 0.1nM [¹²⁵I]MCP-1 in the presence of various concentrations of 1D9 or theisotype-matched anti-CXCR3 antibody 1C6. The amount of bound tracer wasdetermined by separation of free from bound by filtration andscintillation counting. The data was analyzed to determine the IC₅₀value by non-linear regression using a 4-parameter logistic equationwith KaleidaGraph software.

FIG. 5 illustrates that mAb 1D9 inhibits [¹²⁵I]MCP-1 binding to freshhuman PBMC. Freshly isolated peripheral blood mononuclear cells(500,000) were incubated with 0.1 nM [¹²⁵I]MCP-1 in the presence ofvarious concentrations of 1D9 or the isotype-matched anti-CXCR3 antibody1C6. The amount of bound tracer was determined by separation of freefrom bound by filtration and scintillation counting. The data wasanalyzed to determine the IC₅₀ value as for FIG. 4.

FIGS. 6A and 6B are graphs demonstrating that mAb 1D9 inhibitsMCP-1-induced chemotaxis, but not RANTES-induced chemotaxis, of freshPBMC.

FIG. 6A shows the results of chemotaxis assays of PBMC to 10 nM MCP-1with no antibody, or 0.1 or 10 μg/ml of 1D9 or nonspecific murine IgG2a.The spontaneous nonspecific migration is also indicated.

FIG. 6B shows the results of chemotaxis assays of PBMC to 10 nM RANTESwith no antibody, 10 μg/ml 1D9 or 10 μg/ml nonspecific murine IgG2a. Thespontaneous nonspecific migration in the absence of RANTES is alsoindicated.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to an antibody (anti-CCR2) or functionalfragment thereof which binds mammalian CC-chemokine receptor 2 (CCR2,CKR-2, MCP-1RA or MCP-1RB) or a portion of CCR2. In one embodiment, theantibody has specificity for human or rhesus CCR2 or portion thereof. Inone embodiment, the antibodies (immunoglobulins) are raised against anisolated and/or recombinant mammalian CCR2 or portion thereof (e.g.,peptide) or against a host cell which expresses mammalian CCR2. In apreferred embodiment, the antibodies specifically bind human CCR2receptor(s) (e.g., CCR2a and/or CCR2b) or a portion thereof, and in aparticularly preferred embodiment the antibodies have specificity for anaturally occurring or endogenous human CCR2. As used herein,“CC-chemokine receptor 2” (“CCR2”) refers to CC-chemokine receptor 2aand/or CC-chemokine receptor 2b. Antibodies or functional fragmentsthereof which can inhibit one or more functions characteristic of amammalian CCR2, such as a binding activity (e.g., ligand, inhibitorand/or promoter binding), a signaling activity (e.g., activation of amammalian G protein, induction of a rapid and transient increase in theconcentration of cytosolic free calcium [Ca²⁺]i), and/or stimulation ofa cellular response (e.g., stimulation of chemotaxis, exocytosis orinflammatory mediator release by leukocytes, integrin activation) arealso encompassed by the present invention, such as an antibody which caninhibit binding of a ligand (i.e., one or more ligands) to CCR2 and/orone or more functions mediated by CCR2 in response to a ligand. Forexample, in one aspect, the antibodies or functional fragments thereofcan inhibit (reduce or prevent) the interaction of receptor with anatural ligand, such as MCP-1, MCP-2, MCP-3 and/or MCP-4. In anotheraspect, an antibody or functional fragment thereof that binds to CCR2can inhibit binding of MCP-1, MCP-2, MCP-3 and/or MCP-4 and/or HIV tomammalian CCR2 (e.g., human CCR2, non-human primate CCR2, murine CCR2).The antibodies or functional fragments thereof of the present inventioncan inhibit functions mediated by human CCR2, including leukocytetrafficking, HIV entry into a cell, T cell activation, inflammatorymediator release and/or leukocyte degranulation. Preferably, theantibodies or fragments can bind CCR2 with an affinity of at least about0.1×10⁻⁹ M, preferably at least about 1×10⁻⁹ M, and more preferably atleast about 3×10⁻⁹ M. In a particular embodiment, antibodies orfunctional fragments thereof demonstrate inhibition of chemokine-induced(e.g., MCP-1-induced) chemotaxis of cells (e.g., PBMC) at less thanabout 150 μg/ml, preferably less than about 100 μg/ml, more preferablyless than about 50 μg/ml, and even more preferably less than about 20μg/ml.

In a further embodiment of the invention, the antibodies or functionalfragments thereof of the invention can inhibit binding of a CCR2 ligand(e.g., a chemokine) to CCR2 with an IC₅₀ of less than about 1.0 μg/ml,preferably less than about 0.05 μg/ml, and more preferably less thanabout 0.005 μg/ml.

Murine monoclonal antibodies specific for CCR2, designated 1D9 and 8G2,were produced as described herein. In a preferred embodiment, theantibodies of the present invention bind human CCR2, and have anepitopic specificity which is the same as or similar to that of murine1D9 or 8G2 antibody described herein. Antibodies with an epitopicspecificity which is the same as or similar to that of murine 1D9monoclonal antibody can be identified by their ability to compete withmurine 1D9 monoclonal antibody for binding to human CCR2 (e.g., to cellsbearing human CCR2, such as transfectants bearing CCR2, CD8+ cells, CD4+cells, CDR45RO+ cells, CD25+ cells, monocytes, dendritic cells,macrophages and basophils). Similarly, antibodies with an epitopicspecificity which is the same as or similar to that of murine 8G2monoclonal antibody can be identified by their ability to compete withmurine 8G2 monoclonal antibody for binding to human CCR2. Using receptorchimeras (Rucker et al., Cell 87:437-446 (1996)), the binding site ofmAbs 1D9 and 8G2 has been mapped to the amino-terminal domain of humanCC-chemokine receptor 2, specifically to an epitope comprising fromabout amino acid 1 to about amino acid 30 of the protein. Using these orother suitable techniques, antibodies having an epitopic specificitywhich is the same as or similar to that of an antibody of the presentinvention can be identified. mAbs 1D9 and 8G2 have epitopic specificityfor the amino-terminal domain of the CCR2 receptor, e.g., from aboutamino acid number 1 to about amino acid number 30 of the receptorprotein. Thus, the invention pertains to an antibody or functionalportion thereof which binds to the amino-terminal domain or portionthereof of mammalian CC-chemokine receptor 2, and particularly to anepitope comprising from about amino acid 1 to about amino acid 30 ofmammalian CC-chemokine receptor 2.

The invention also relates to a bispecific antibody, or functionalfragment thereof (e.g., F(ab′)₂), which has the same or similar epitopicspecificity as at least two of the antibodies described herein (see,e.g., U.S. Pat. No. 5,141,736 (Iwasa et al.), U.S. Pat. Nos. 4,444,878,5,292,668, 5,523,210 (all to Paulus et al.) and U.S. Pat. No. 5,496,549(Yamazaki et al.). For example, a bispecific antibody of the presentinvention can have the same or similar epitopic specificity as mAb 1D9and 8G2, e.g., binds the amino terminal domain, or portion thereof, ofmammalian CCR2 protein.

Hybridoma cell lines producing antibodies according to the presentinvention were deposited on Jul. 17, 1998, on behalf of LeukoSite, Inc.,215 First Street, Cambridge, Mass. 02142, U.S.A., at the American TypeCulture Collection, 10801 University Boulevard, Manassas, Va. 20110,U.S.A., under Accession Nos. HB-12549 (1D9) and HB-12550 (8G2). Thepresent invention also pertains to the hybridoma cell lines depositedunder ATCC Accession No. HB-12549 and ATCC Accession No. HB-12550, aswell as to the monoclonal antibodies produced by the hybridoma celllines deposited under ATCC Accession Nos. HB-12549 and HB-12550.

The antibodies of the present invention can be polyclonal or monoclonal,and the term “antibody” is intended to encompass both polyclonal andmonoclonal antibodies. Furthermore, it is understood that methodsdescribed herein which utilize 8G2 can also utilize functional fragments(e.g., antigen-binding fragments) of 8G2, antibodies which have the sameor similar epitopic specificity as 8G2, and combinations thereof,optionally in combination with antibodies or fragments having anepitopic specificity which is not the same as or similar to 8G2;similarly, methods described as utilizing 1D9 can also utilizefunctional fragments of 1D9, antibodies which have the same or similarepitopic specificity as 1D9, and combinations thereof, optionally incombination with antibodies or fragments having an epitopic specificitywhich is not the same as or similar to 1D9. Antibodies of the presentinvention can be raised against an appropriate immunogen, such asisolated and/or recombinant mammalian CCR2 protein or portion thereof,or synthetic molecules, such as synthetic peptides. In a preferredembodiment, cells which express receptor, such as transfected cells, canbe used as immunogens or in a screen for antibody which binds receptor.

The antibodies of the present invention, and fragments thereof, areuseful in therapeutic, diagnostic and research applications as describedherein. The present invention encompasses an antibody or functionalportion thereof of the present invention (e.g., mAb 1D9 or 8G2, orantigen-binding fragments thereof) for use in therapy (includingprophylaxis) or diagnosis (e.g., of particular diseases or conditions asdescribed herein), and use of such antibodies or functional portionsthereof for the manufacture of a medicament for use in treatment ofdiseases or conditions as described herein.

Preparation of immunizing antigen, and polyclonal and monoclonalantibody production can be performed as described herein, or using othersuitable techniques. A variety of methods have been described (see e.g.,Kohler et al., Nature, 256: 495-497 (1975) and Eur. J. Immunol. 6:511-519 (1976); Milstein et al., Nature 266: 550-552 (1977); Koprowskiet al., U.S. Pat. No. 4,172,124; Harlow, E. and D. Lane, 1988,Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory: ColdSpring Harbor, N.Y.); Current Protocols In Molecular Biology, Vol. 2(Supplement 27, Summer '94), Ausubel, F. M. et al., Eds., (John Wiley &Sons: New York, N.Y.), Chapter 11, (1991)). Generally, a hybridoma canbe produced by fusing a suitable immortal cell line (e.g., a myelomacell line such as SP2/0) with antibody producing cells. The antibodyproducing cell, preferably those of the spleen or lymph nodes, areobtained from animals immunized with the antigen of interest. The fusedcells (hybridomas) can be isolated using selective culture conditions,and cloned by limiting dilution. Cells which produce antibodies with thedesired binding properties can be selected by a suitable assay (e.g.,ELISA).

Other suitable methods of producing or isolating antibodies which bindCCR2, including human or artificial antibodies, can be used, including,for example, methods which select recombinant antibody (e.g., singlechain Fv or Fab) from a library, or which rely upon immunization oftransgenic animals (e.g., mice) capable of producing a repertoire ofhuman antibodies (see e.g., Jakobovits et al., Proc. Natl. Acad. Sci.USA, 90: 2551-2555 (1993); Jakobovits et al., Nature, 362: 255-258(1993); Lonberg et al., U.S. Pat. No. 5,545,806; Surani et al., U.S.Pat. No. 5,545,807).

Single chain antibodies, and chimeric, humanized or primatized(CDR-grafted) antibodies, as well as chimeric or CDR-grafted singlechain antibodies, and the like, comprising portions derived fromdifferent species, are also encompassed by the present invention and theterm “antibody”. The various portions of these antibodies can be joinedtogether chemically by conventional techniques, or can be prepared as acontiguous protein using genetic engineering techniques. For example,nucleic acids encoding a chimeric or humanized chain can be expressed toproduce a contiguous protein. See, e.g., Cabilly et al., U.S. Pat. No.4,816,567; Cabilly et al., European Patent No. 0,125,023 B1; Boss etal., U.S. Pat. No. 4,816,397; Boss et al., European Patent No. 0,120,694B1; Neuberger, M. S. et al., WO 86/01533; Neuberger, M. S. et al.,European Patent No. 0,194,276 B1; Winter, U.S. Pat. No. 5,225,539;Winter, European Patent No. 0,239,400 B1; and Queen et al., U.S. Pat.Nos. 5,585,089, 5,698,761 and 5,698,762. See also, Newman, R. et al.,BioTechnology, 10: 1455-1460 (1992), regarding primatized antibody, andLadner et al., U.S. Pat. No. 4,946,778 and Bird, R. E. et al., Science,242: 423-426 (1988)) regarding single chain antibodies.

In addition, functional fragments of antibodies, including fragments ofchimeric, humanized, primatized or single chain antibodies, can also beproduced. Functional fragments of the foregoing antibodies retain atleast one binding function and/or modulation function of the full-lengthantibody from which they are derived. Preferred functional fragmentsretain an antigen-binding function of a corresponding full-lengthantibody (e.g., the ability to bind a mammalian CCR2). Particularlypreferred functional fragments retain the ability to inhibit one or morefunctions characteristic of a mammalian CCR2, such as a bindingactivity, a signaling activity, and/or stimulation of a cellularresponse. For example, in one embodiment, a functional fragment caninhibit the interaction of CCR2 with one or more of its ligands (e.g.,MCP-1, MCP-2, MCP-3 and/or MCP-4) and/or can inhibit one or morereceptor-mediated functions, such as leukocyte trafficking, HIV entryinto cells, T cell activation, inflammatory mediator release and/orleukocyte degranulation.

For example, antibody fragments capable of binding to a mammalian CCR2receptor or portion thereof, including, but not limited to, Fv, Fab,Fab′ and F(ab′)₂ fragments are encompassed by the invention. Suchfragments can be produced by enzymatic cleavage or by recombinanttechniques, for example. For instance, papain or pepsin cleavage cangenerate Fab or F(ab′)₂ fragments, respectively. Antibodies can also beproduced in a variety of truncated forms using antibody genes in whichone or more stop codons has been introduced upstream of the natural stopsite. For example, a chimeric gene encoding a F(ab′)₂ heavy chainportion can be designed to include DNA sequences encoding the CH_(l)domain and hinge region of the heavy chain.

The term “humanized immunoglobulin” as used herein refers to animmunoglobulin comprising portions of immunoglobulins of differentorigin, wherein at least one portion is of human origin. Accordingly,the present invention relates to a humanized immunoglobulin which bindsmammalian CCR2 (e.g., human CCR2, murine CCR2), said immunoglobulincomprising an antigen-binding region of nonhuman origin (e.g., rodent)and at least a portion of an immunoglobulin of human origin (e.g., ahuman framework region, a human constant region or portion thereof). Forexample, the humanized antibody can comprise portions derived from animmunoglobulin of nonhuman origin with the requisite specificity, suchas a mouse, and from immunoglobulin sequences of human origin (e.g., achimeric immunoglobulin), joined together chemically by conventionaltechniques (e.g., synthetic) or prepared as a contiguous polypeptideusing genetic engineering techniques (e.g., DNA encoding the proteinportions of the chimeric antibody can be expressed to produce acontiguous polypeptide chain). Another example of a humanizedimmunoglobulin of the present invention is an immunoglobulin containingone or more immunoglobulin chains comprising a CDR of nonhuman origin(e.g., one or more CDRs derived from an antibody of nonhuman origin) anda framework region derived from a light and/or heavy chain of humanorigin (e.g., CDR-grafted antibodies with or without framework changes).In one embodiment, the humanized immunoglobulin can compete with murine1D9 or 8G2 monoclonal antibody for binding to human CCR2. In a preferredembodiment, the antigen-binding region of the humanized immunoglobulin(a) is derived from 1D9 monoclonal antibody (e.g., as in a humanizedimmunoglobulin comprising CDR1, CDR2 and CDR3 of the 1D9 light chain andCDR1, CDR2 and CDR3 of the 1D9 heavy chain) or (b) is derived from 8G2monoclonal antibody (e.g., as in a humanized immunoglobulin comprisingCDR1, CDR2 and CDR3 of the 8G2 light chain and CDR1, CDR2 and CDR3 ofthe 8G2 heavy chain). Chimeric or CDR-grafted single chain antibodiesare also encompassed by the term humanized immunoglobulin.

Such humanized immunoglobulins can be produced using synthetic and/orrecombinant nucleic acids to prepare genes (e.g., cDNA) encoding thedesired humanized chain. For example, nucleic acid (e.g., DNA) sequencescoding for humanized variable regions can be constructed using PCRmutagenesis methods to alter DNA sequences encoding a human or humanizedchain, such as a DNA template from a previously humanized variableregion (see e.g., Kamman, M., et al., Nucl. Acids Res., 17: 5404(1989)); Sato, K., et al., Cancer Research, 53: 851-856 (1993);Daugherty, B. L. et al., Nucleic Acids Res., 19(9): 2471-2476 (1991);and Lewis, A. P. and J. S. Crowe, Gene, 101: 297-302 (1991)). Usingthese or other suitable methods, variants can also be readily produced.In one embodiment, cloned variable regions can be mutagenized, andsequences encoding variants with the desired specificity can be selected(e.g., from a phage library; see e.g., Krebber et al., U.S. Pat. No.5,514,548; Hoogenboom et al., WO 93/06213, published Apr. 1, 1993)).

Anti-idiotypic antibodies are also provided. Anti-idiotypic antibodiesrecognize antigenic determinants associated with the antigen-bindingsite of another antibody. Anti-idiotypic antibodies can be preparedagainst second antibody by immunizing an animal of the same species, andpreferably of the same strain, as the animal used to produce the secondantibody. See e.g., U.S. Pat. No. 4,699,880.

The present invention also pertains to the hybridoma cell linesdeposited under ATCC Accession Nos. HB-12549 and HB-12550, as well as tothe monoclonal antibodies produced by the hybridoma cell lines depositedunder ATCC Accession Nos. HB-12549 and HB-12550. The cell lines of thepresent invention have uses other than for the production of themonoclonal antibodies. For example, the cell lines of the presentinvention can be fused with other cells (such as suitably drug-markedhuman myeloma, mouse myeloma, human-mouse heteromyeloma or humanlymphoblastoid cells) to produce additional hybridomas, and thus providefor the transfer of the genes encoding the monoclonal antibodies. Inaddition, the cell lines can be used as a source of nucleic acidsencoding the anti-CCR2 immunoglobulin chains, which can be isolated andexpressed (e.g., upon transfer to other cells using any suitabletechnique (see e.g., Cabilly et al., U.S. Pat. No. 4,816,567; Winter,U.S. Pat. No. 5,225,539)). For instance, clones comprising a rearrangedanti-CCR2 light or heavy chain can be isolated (e.g., by PCR) or cDNAlibraries can be prepared from mRNA isolated from the cell lines, andcDNA clones encoding an anti-CCR2 immunoglobulin chain can be isolated.Thus, nucleic acids encoding the heavy and/or light chains of theantibodies or portions thereof can be obtained and used in accordancewith recombinant DNA techniques for the production of the specificimmunoglobulin, immunoglobulin chain, or variants thereof (e.g.,humanized immunoglobulins) in a variety of host cells or in an in vitrotranslation system. For example, the nucleic acids, including cDNAs, orderivatives thereof encoding variants such as a humanized immunoglobulinor immunoglobulin chain, can be placed into suitable prokaryotic oreukaryotic vectors (e.g., expression vectors) and introduced into asuitable host cell by an appropriate method (e.g., transformation,transfection, electroporation, infection), such that the nucleic acid isoperably linked to one or more expression control elements (e.g., in thevector or integrated into the host cell genome). For production, hostcells can be maintained under conditions suitable for expression (e.g.,in the presence of inducer, suitable media supplemented with appropriatesalts, growth factors, antibiotic, nutritional supplements, etc.),whereby the encoded polypeptide is produced. If desired, the encodedprotein can be recovered and/or isolated (e.g., from the host cells,medium, milk). It will be appreciated that the method of productionencompasses expression in a host cell of a transgenic animal (see e.g.,WO 92/03918, GenPharm International, published Mar. 19, 1992).

As described herein, antibodies and functional fragments thereof of thepresent invention can block (inhibit) binding of a ligand to CCR2 and/orinhibit function associated with binding of the ligand to the CCR2. Asdiscussed below various methods can be used to assess inhibition ofbinding of a ligand to CCR2 and/or function associated with binding ofthe ligand to the receptor.

Binding Assays

As used herein “mammalian CCR2 protein” refers to naturally occurring orendogenous mammalian CCR2 proteins and to proteins having an amino acidsequence which is the same as that of a naturally occurring orendogenous corresponding mammalian CCR2 protein (e.g., recombinantproteins). Accordingly, as defined herein, the term includes maturereceptor protein, polymorphic or allelic variants, and other isoforms ofa mammalian CCR2 (e.g., produced by alternative splicing or othercellular processes), and modified or unmodified forms of the foregoing(e.g., glycosylated, unglycosylated). Mammalian CCR2 proteins can beisolated and/or recombinant proteins (including synthetically producedproteins). Naturally occurring or endogenous mammalian CCR2 proteinsinclude wild type proteins such as mature CCR2, polymorphic or allelicvariants and other isoforms which occur naturally in mammals (e.g.,humans, non-human primates), such as the CCR2 a and CCR2 b forms of thereceptor protein which are produced by alternative splicing of thecarboxy-terminus of the protein. Such proteins can be recovered orisolated from a source which naturally produces mammalian CCR2, forexample. These proteins and mammalian CCR2 proteins having the sameamino acid sequence as a naturally occurring or endogenous correspondingmammalian CCR2, are referred to by the name of the corresponding mammal.For example, where the corresponding mammal is a human, the protein isdesignated as a human CCR2 protein (e.g., a recombinant human CCR2produced in a suitable host cell).

“Functional variants” of mammalian CCR2 proteins include functionalfragments, functional mutant proteins, and/or functional fusion proteins(e.g., produced via mutagenesis and/or recombinant techniques).Generally, fragments or portions of mammalian CCR2 proteins includethose having a deletion (i.e., one or more deletions) of an amino acid(i.e., one or more amino acids) relative to the mature mammalian CCR2protein (such as N-terminal, C-terminal or internal deletions).Fragments or portions in which only contiguous amino acids have beendeleted or in which non-contiguous amino acids have been deletedrelative to mature mammalian CCR2 protein are also envisioned.

Generally, mutants of mammalian CCR2 proteins include natural orartificial variants of a mammalian CCR2 protein differing by theaddition, deletion and/or substitution of one or more contiguous ornon-contiguous amino acid residues (e.g., receptor chimeras). Suchmutations can be in a conserved region or nonconserved region (comparedto other CXC and/or CC chemokine receptors), extracellular, cytoplasmic,or transmembrane region, for example.

Generally, fusion proteins encompass polypeptides comprising a mammalianCCR2 (e.g., human CCR2) as a first moiety, linked via a peptide cond toa second moiety not occurring in the mammalian CCR2 as found in nature.Thus, the second moiety can be an amino acid, oligopeptide orpolypeptide. The first moiety can be in an N-terminal location,C-terminal location or internal to the fusion protein. In oneembodiment, the fusion protein comprises an affinity ligand (e.g., anenzyme, an antigen, epitope tage) as the first moiety, and a secondmoiety comprising a linker sequence and human CCR2 or a portion thereof.

A “functional fragment or portion”, “functional mutant” and/or“functional fusion protein” of a mammalian CCR2 protein refers to anisolated and/or recombinant protein or polypeptide which has at leastone function characteristic of a mammalian CCR2 protein as describedherein, such as a binding activity, a signaling activity and/or abilityto stimulate a cellular response. Preferred functional variants can binda ligand (i.e., one or more ligands such as MCP-1, MCP-2, MCP-3 and/orMCP-4), and are referred to herein as “ligand binding variants”.

In one embodiment, a functional variant of mammalian CCR2 shares atleast about 85% sequence identity with said mammalian CCR2, preferablyat least about 90% sequence identity, and more preferably at least about95% sequence identity with said mammalian CCR2. The nucleic acid andamino acid sequences of human CCR2a and CCR2b are described in U.S. Pat.No. 5,707,815. Sequence identity can be determine using a suitableprogram, such as the Blastx program (Version 1.4), using appropriateparameters, such as default parameters. In one embodiment, parametersfor Blastx search are scoring matrix BLOSUM62, W=3. In anotherembodiment, a functional variant comprises a nucleic acid sequence whichis different from the naturally-occurring nucleic acid molecule butwhich, due to the degeneracy of the genetic code, encodes mammalian CCR2or a portion thereof.

A composition comprising an isolated and/or recombinant mammalian CCR2or functional variant thereof can be maintained under conditionssuitable for binding, the mammalian CCR2 or variant is contacted with anantibody or fragment to be tested, and binding is detected or measureddirectly or indirectly. In one embodiment, cells which naturally expressCCR2 or cells comprising a recombinant nucleic acid sequence whichencodes a mammalian CCR2 or variant thereof are used. The cells aremaintained under conditions appropriate for expression of receptor. Thecells are contacted with an antibody or fragment under conditionssuitable for binding (e.g., in a suitable binding buffer), and bindingis detected by standard techniques. To determine binding, the extent ofbinding an be determined relative to a suitable control (e.g., comparedwith background determined in the absence of antibody, compared withbinding of a second antibody (i.e., a standard), compared with bindingof antibody to untransfected cells). A cellular fraction, such as amembrane fraction, containing receptor or liposomes comprising receptorcan be used in lieu of whole cells.

In one embodiment, the antibody is labeled with a suitable label (e.g.,fluorescent label, isotope label, antigen or epitope label, enzymelabel), and binding is determined by detection of the label. In anotherembodiment, bound antibody can be detected by labeled second antibody.Specificity of binding can be assessed by competition or displacement,for example, using unlabeled antibody or a ligand as competitor.

Binding inhibition assays can also be used to identify antibodies orfragments thereof which bind CCR2 and inhibit binding of anothercompound such as a ligand (e.g., MCP-1, MCP-2, MCP-3 and/or MCP-4) toCCR2 or a functional variant. For example, a binding assay can beconducted in which a reduction in the binding of a ligand of CCR2 (inthe presence of an antibody), as compared to binding of the ligand inthe absence of the antibody, is detected or measured. A compositioncomprising an isolated and/or recombinant mammalian CCR2 or functionalvariant thereof can be contacted with the ligand and antibodysimultaneously, or one after the other, in either order. A reduction inthe extent of binding of the ligand in the presence of the antibody, isindicative of inhibition of binding by the antibody. For example,binding of the ligand could be decreased or abolished.

In one embodiment, direct inhibition of the binding of a ligand (e.g., achemokine such as MCP-1) to a mammalian CCR2 or variant thereof by anantibody or fragment is monitored. For example, the ability of anantibody to inhibit the binding of ¹²⁵I-labeled MCP-1, ¹²⁵I-labeledMCP-2, ¹²⁵I-labeled MCP-3 or ¹²⁵I-labeled MCP-4 to mammalian CCR2 can bemonitored. Such an assay can be conducted using suitable cells bearingCCR2 or a functional variant thereof, such as isolated blood cells(e.g., T cells, PBMC) or a suitable cell line naturally expressing CCR2,or a cell line containing nucleic acid encoding a mammalian CCR2, or amembrane fraction from said cells, for instance.

Other methods of identifying the presence of an antibody which bindsCCR2 are available, such as other suitable binding assays, or methodswhich monitor events which are triggered by receptor binding, includingsignaling function and/or stimulation of a cellular response (e.g.,leukocyte trafficking).

It will be understood that the inhibitory effect of antibodies of thepresent invention can be assessed in a binding inhibition assay.Competition between antibodies for receptor binding can also be assessedin the method. Antibodies which are identified in this manner can befurther assessed to determine whether, subsequent to binding, they actto inhibit other functions of CCR2 and/or to assess their therapeuticutility.

Signaling Assays

The binding of a ligand or promoter, such as an agonist, to CCR2 canresult in signaling by this G protein-coupled receptor, and the activityof G proteins as well as other intracellular signaling molecules isstimulated. The induction of signaling function by a compound (e.g., anantibody or fragment thereof) can be monitored using any suitablemethod. Such an assay can be used to identify antibody agonists of CCR2.The inhibitory activity of an antibody or functional fragment thereofcan be determined using a ligand or promoter in the assay, and assessingthe ability of the antibody to inhibit the activity induced by ligand orpromoter.

G protein activity, such as hydrolysis of GTP to GDP, or later signalingevents triggered by receptor binding, such as induction of rapid andtransient increase in the concentration of intracellular (cytosolic)free calcium [Ca²⁺]i, can be assayed by methods known in the art orother suitable methods (see e.g., Neote, K. et al., Cell, 72: 415-4251993); Van Riper et al., J. Exp. Med., 177: 851-856 (1993); Dahinden, C.A. et al., J. Exp. Med., 179: 751-756 (1994)).

For example, the functional assay of Sledziewski et al. using hybrid Gprotein coupled receptors can be used to monitor the ability a ligand orpromoter to bind receptor and activate a G protein (Sledziewski et al.,U.S. Pat. No. 5,284,746, the teachings of which are incorporated hereinby reference).

Such assays can be performed in the presence of the antibody or fragmentthereof to be assessed, and the ability of the antibody or fragment toinhibit the activity induced by the ligand or promoter is determinedusing known methods and/or methods described herein.

Chemotaxis and Assays of Cellular Stimulation

Chemotaxis assays can also be used to assess the ability of an antibodyor functional fragment thereof to block binding of a ligand to mammalianCCR2 or functional variant thereof and/or inhibit function associatedwith binding of the ligand to the receptor. These assays are based onthe functional migration of cells in vitro or in vivo induced by acompound. Chemotaxis can be assessed as described in the Examples, e.g.,in an assay utilizing a 96-well chemotaxis plate, or using otherart-recognized methods for assessing chemotaxis. For example, the use ofan in vitro transendothelial chemotaxis assay is described by Springeret al. (Springer et al., WO 94/202, published Sep. 15, 1994, theteachings of which are incorporated herein by reference; see also Bermanet al., Immunol. Invest. 17: 625-677 (1988)). Migration acrossendothelium into collagen gels has also been described (Kavanaugh etal., J. Immunol., 146: 4149-4156 (1991)). Stable transfectants of mouseL1-2 pre-B cells or of other suitable host cells capable of chemotaxiscan be used in chemotaxis assays, for example.

Generally, chemotaxis assays monitor the directional movement ormigration of a suitable cell (such as a leukocyte (e.g., lymphocyte,eosinophil, basophil)) into or through a barrier (e.g., endothelium, afilter), toward increased levels of a compound, from a first surface ofthe barrier toward an opposite second surface. Membranes or filtersprovide convenient barriers, such that the directional movement ormigration of a suitable cell into or through a filter, toward increasedlevels of a compound, from a first surface of the filter toward anopposite second surface of the filter, is monitored. In some assays, themembrane is coated with a substance to facilitate adhesion, such asICAM-1, fibronectin or collagen. Such assays provide an in vitroapproximation of leukocyte “homing”.

For example, one can detect or measure inhibition of the migration ofcells in a suitable container (a containing means), from a first chamberinto or through a microporous membrane into a second chamber whichcontains an antibody to be tested, and which is divided from the firstchamber by the membrane. A suitable membrane, having a suitable poresize for monitoring specific migration in response to compound,including, for example, nitrocellulose, polycarbonate, is selected. Forexample, pore sizes of about 3-8 microns, and preferably about 5-8microns can be used. Pore size can be uniform on a filter or within arange of suitable pore sizes.

To assess migration and inhibition of migration, the distance ofmigration into the filter, the number of cells crossing the filter thatremain adherent to the second surface of the filter, and/or the numberof cells that accumulate in the second chamber can be determined usingstandard techniques (e.g., microscopy). In one embodiment, the cells arelabeled with a detectable label (e.g., radioisotope, fluorescent label,antigen or epitope label), and migration can be assessed in the presenceand absence of the antibody or fragment by determining the presence ofthe label adherent to the membrane and/or present in the second chamberusing an appropriate method (e.g., by detecting radioactivity,fluorescence, immunoassay). The extent of migration induced by anantibody agonist can be determined relative to a suitable control (e.g.,compared to background migration determined in the absence of theantibody, compared to the extent of migration induced by a secondcompound (i.e., a standard), compared with migration of untransfectedcells induced by the antibody).

In one embodiment, particularly for T cells, monocytes or cellsexpressing a mammalian CCR2, transendothelial migration can bemonitored. In this embodiment, transmigration through an endothelialcell layer is assessed. To prepare the cell layer, endothelial cells canbe cultured on a microporous filter or membrane, optionally coated witha substance such as collagen, fibronectin, or other extracellular matrixproteins, to facilitate the attachment of endothelial cells. Preferably,endothelial cells are cultured until a confluent monolayer is formed. Avariety of mammalian endothelial cells can are available for monolayerformation, including for example, vein, artery or microvascularendothelium, such as human umbilical vein endothelial cells (CloneticsCorp, San Diego, Calif.). To assay chemotaxis in response to aparticular mammalian receptor, endothelial cells of the same mammal arepreferred; however endothelial cells from a heterologous mammalianspecies or genus can also be used.

Generally, the assay is performed by detecting the directional migrationof cells into or through a membrane or filter, in a direction towardincreased levels of a compound, from a first surface of the filtertoward an opposite second surface of the filter, wherein the filtercontains an endothelial cell layer on a first surface. Directionalmigration occurs from the area adjacent to the first surface, into orthrough the membrane, towards a compound situated on the opposite sideof the filter. The concentration of compound present in the areaadjacent to the second surface, is greater than that in the areaadjacent to the first surface.

In one embodiment used to test for an antibody inhibitor, a compositioncomprising cells capable of migration and expressing a mammalian CCR2receptor can be placed in the first chamber. A composition comprisingone or more ligands or promoters capable of inducing chemotaxis of thecells in the first chamber (having chemoattractant function) is placedin the second chamber. Preferably shortly before the cells are placed inthe first chamber, or simultaneously with the cells, a compositioncomprising the antibody to be tested is placed, preferably, in the firstchamber. Antibodies or functional fragments thereof which can bindreceptor and inhibit the induction of chemotaxis, by a ligand orpromoter, of the cells expressing a mammalian CCR2 in this assay areinhibitors of receptor function (e.g., inhibitors of stimulatoryfunction). A reduction in the extent of migration induced by the ligandor promoter in the presence of the antibody or fragment is indicative ofinhibitory activity. Separate binding studies (see above) could beperformed to determine whether inhibition is a result of binding of theantibody to receptor or occurs via a different mechanism.

In vivo assays which monitor leukocyte infiltration of a tissue, inresponse to injection of a compound (e.g., chemokine or antibody) in thetissue, are described below (see Models of Inflammation). These modelsof in vivo homing measure the ability of cells to respond to a ligand orpromoter by emigration and chemotaxis to a site of inflammation and toassess the ability of an antibody or fragment thereof to block thisemigration.

In addition to the methods described, the effects of an antibody orfragment on the stimulatory function of CCR2 can be assessed bymonitoring cellular responses induced by active receptor, using suitablehost cells containing receptor.

Identification of Additional Ligands, Inhibitors and/or Promoters ofMammalian CCR2 Function

The assays described above, which can be used to assess binding andfunction of the antibodies and fragments of the present invention, canbe adapted to identify additional ligands or other substances which binda mammalian CCR2 or functional variant thereof, as well as inhibitorsand/or promoters of mammalian CCR2 function. For example, agents havingthe same or a similar binding specificity as that of an antibody of thepresent invention or functional portion thereof can be identified by acompetition assay with said antibody or portion thereof. Thus, thepresent invention also encompasses methods of identifying ligands of thereceptor or other substances which bind a mammalian CCR2 protein, aswell as inhibitors (e.g., antagonists) or promoters (e.g., agonists) ofreceptor function. In one embodiment, cells bearing a mammalian CCR2protein or functional variant thereof (e.g., leukocytes, cell lines orsuitable host cells which have been engineered to express a mammalianCCR2 protein or functional variant encoded by a nucleic acid introducedinto said cells) are used in an assay to identify and assess theefficacy of ligands or other substances which bind receptor, includinginhibitors or promoters of receptor function. Such cells are also usefulin assessing the function of the expressed receptor protein orpolypeptide.

According to the present invention, ligands and other substances whichbind receptor, inhibitors and promoters of receptor function can beidentified in a suitable assay, and further assessed for therapeuticeffect. Inhibitors of receptor function can be used to inhibit (reduceor prevent) receptor activity, and ligands and/or promoters can be usedto induce (trigger or enhance) normal receptor function where indicated.Thus, the present invention provides a method of treating inflammatorydiseases, including autoimmune disease and graft rejection, comprisingadministering an inhibitor of receptor function to an individual (e.g.,a mammal). The present invention further provides a method ofstimulating receptor function by administering a novel ligand orpromoter of receptor function to an individual, providing a new approachto selective stimulation of leukocyte function, which is useful, forexample, in the treatment of infectious diseases and cancer.

As used herein, a “ligand” of a mammalian CCR2 protein refers to aparticular class of substances which bind to a mammalian CCR2 protein,including natural ligands and synthetic and/or recombinant forms ofnatural ligands. Infectious agents having a tropism for mammalian CCR2positive cells (e.g., viruses such as HIV) can also bind to a mammalianCCR2 protein. A natural ligand of a selected mammalian receptor is of amammalian origin which is the same as that of the mammalian CCR2 protein(e.g., a chemokine such as MCP-1, MCP-2, MCP-3 and/or MCP-4). In apreferred embodiment, ligand binding of a mammalian CCR2 protein occurswith high affinity.

As used herein, an “inhibitor” is a substance which inhibits (decreasesor prevents) at least one function characteristic of a mammalian CCR2protein (e.g., a human CCR2), such as a binding activity (e.g., ligandbinding, promoter binding, antibody binding), a signaling activity(e.g., activation of a mammalian G protein, induction of rapid andtransient increase in the concentration of cytosolic free calcium[Ca²⁺]i), and/or cellular response function (e.g., stimulation ofchemotaxis, exocytosis or inflammatory mediator release by leukocytes).An inhibitor is also a substance which inhibits HIV entry into a cell.The term inhibitor refers to substances including antagonists which bindreceptor (e.g., an antibody, a mutant of a natural ligand, smallmolecular weight organic molecules, other competitive inhibitors ofligand binding), and substances which inhibit receptor function withoutbinding thereto (e.g., an anti-idiotypic antibody).

As used herein, a “promoter” is a substance which promotes (induces,causes, enhances or increases) at least one function characteristic of amammalian CCR2 protein (e.g., a human CCR2), such as a binding activity(e.g., ligand, inhibitor and/or promoter binding), a signaling activity(e.g., activation of a mammalian G protein, induction of rapid andtransient increase in the concentration of cytosolic free calcium[Ca²⁺]i), and/or a cellular response function (e.g., stimulation ofchemotaxis, exocytosis or inflammatory mediator release by leukocytes).The term promoter refers to substances including agonists which bindreceptor (e.g., an antibody, a homolog of a natural ligand from anotherspecies), and substances which promote receptor function without bindingthereto (e.g., by activating an associated protein). In a preferredembodiment, the agonist is other than a homolog of a natural ligand.

Thus, the invention also relates to a method of detecting or identifyingan agent which binds a mammalian CC-chemokine receptor 2 or ligandbinding variant thereof, including ligands, inhibitors, promoters, andother substances which bind a mammalian CCR2 receptor or functionalvariant. According to the method, an agent to be tested, an antibody orantigen-binding fragment of the present invention (e.g., 8G2, 1D9, anantibody having an epitopic specificity which is the same as or similarto that of 8G2 or 1D9, and antigen-binding fragments thereof) and acomposition comprising a mammalian CC-chemokine receptor 2 or a ligandbinding variant thereof can be combined. The foregoing components arecombined under conditions suitable for binding of the antibody orantigen-binding fragment to mammalian CC-chemokine receptor 2 or aligand binding variant thereof, and binding of the antibody or fragmentto the mammalian CC-chemokine receptor 2 or ligand binding variant isdetected or measured, either directly or indirectly, according tomethods described herein or other suitable methods. A decrease in theamount of complex formed relative to a suitable control (e.g., in theabsence of the agent to be tested) is indicative that the agent bindssaid receptor or variant. The composition comprising a mammalianCC-chemokine receptor 2 or a ligand binding variant thereof can be amembrane fraction of a cell bearing recombinant chemokine receptor 2protein or ligand binding variant thereof. The antibody or fragmentthereof can be labeled with a label such as a radioisotope, spin label,antigen or epitope label, enzyme label, fluorescent group andchemiluminescent group.

In one embodiment, the invention relates to a method of detecting oridentifying an agent which binds a mammalian CC-chemokine receptor 2 ora ligand binding variant thereof, comprising combining an agent to betested, an antibody or antigen-binding fragment of the present invention(e.g., 1D9, 8G2, an antibody having an epitopic specificity which is thesame as or similar to that of 1D9 or 8G2, or antigen-binding fragmentsthereof) and a cell bearing a mammalian CC-chemokine receptor 2 or aligand binding variant thereof. The foregoing components are combinedunder conditions suitable for binding of the antibody or antigen-bindingfragment to the CCR2 protein or ligand binding variant thereof, andbinding of the antibody or fragment to the mammalian CC-chemokinereceptor 2 or variant is detected or measured, either directly orindirectly, by methods described herein and or other suitable methods. Adecrease in the amount of complex formed relative to a suitable controlis indicative that the agent binds the receptor or variant. The antibodyor fragment thereof can be labeled with a label selected from the groupconsisting of a radioisotope, spin label, antigen or epitope label,enzyme label, fluorescent group and chemiluminescent group. These andsimilar assays can be used to detect agents, including ligands (e.g.,chemokines or strains of HIV which interact with CCR2) or othersubstances, including inhibitors or promoters of receptor function,which can bind CCR2 and compete with the antibodies described herein forbinding to the receptor.

The assays described above can be used, alone or in combination witheach other or other suitable methods, to identify ligands or othersubstances which bind a mammalian CCR2 protein, and inhibitors orpromoters of a mammalian CCR2 protein or variant. The in vitro methodsof the present invention can be adapted for high-throughput screening inwhich large numbers of samples are processed (e.g., a 96-well format).Cells expressing mammalian CCR2 (e.g., human CCR2) at levels suitablefor high-throughput screening can be used, and thus, are particularlyvaluable in the identification and/or isolation of ligands or othersubstances which bind receptor, and inhibitors or promoters of mammalianCCR2 proteins. Expression of receptor can be monitored in a variety ofways. For instance, expression can be monitored using antibodies of thepresent invention which bind receptor or a portion thereof. Also,commercially available antibodies can be used to detect expression of anantigen- or epitope-tagged fusion protein comprising a receptor proteinor polypeptide (e.g., FLAG tagged receptors), and cells expressing thedesired level can be selected.

Nucleic acid encoding a mammalian CCR2 protein or functional variantthereof can be incorporated into an expression system to produce areceptor protein or polypeptide. An isolated and/or recombinantmammalian CCR2 protein or variant, such as a receptor expressed in cellsstably or transiently transfected with a construct comprising arecombinant nucleic acid encoding a mammalian CCR2 protein or variant,or in a cell fraction containing receptor (e.g., a membrane fractionfrom transfected cells, liposomes incorporating receptor), can be usedin tests for receptor function. The receptor can be further purified ifdesired. Testing of receptor function can be carried out in vitro or invivo.

An isolated and/or recombinant mammalian CCR2 protein or functionalvariant thereof, such as a human CCR2, can be used in the presentmethod, in which the effect of a compound is assessed by monitoringreceptor function as described herein or using other suitabletechniques. For example, stable or transient transfectants (e.g.,baculovirus infected Sf9 cells, stable tranfectants of mouse L1/2 pre-Bcells), can be used in binding assays. Stable transfectants of Jurkatcells or of other suitable cells capable of chemotaxis can be used(e.g., mouse L1/2 pre-B cells) in chemotaxis assays, for example.

According to the method of the present invention, compounds can beindividually screened or one or more compounds can be testedsimultaneously according to the methods herein. Where a mixture ofcompounds is tested, the compounds selected by the processes describedcan be separated (as appropriate) and identified by suitable methods(e.g., PCR, sequencing, chromatography, mass spectroscopy). The presenceof one or more compounds (e.g., a ligand, inhibitor, promoter) in a testsample can also be determined according to these methods.

Large combinatorial libraries of compounds (e.g., organic compounds,recombinant or synthetic peptides, “peptoids”, nucleic acids) producedby combinatorial chemical synthesis or other methods can be tested (seee.g., Zuckerman, R. N. et al., J. Med. Chem., 37: 2678-2685 (1994) andreferences cited therein; see also, Ohlmeyer, M. H. J. et al., Proc.Natl. Acad. Sci. USA 90:10922-10926 (1993) and DeWitt, S. H. et al.,Proc. Natl. Acad. Sci. USA 90:6909-6913 (1993), relating to taggedcompounds; Rutter, W. J. et al. U.S. Pat. No. 5,010,175; Huebner, V. D.et al., U.S. Pat. No. 5,182,366; and Geysen, H. M., U.S. Pat. No.4,833,092). Where compounds selected from a combinatorial library by thepresent method carry unique tags, identification of individual compoundsby chromatographic methods is possible.

In one embodiment, phage display methodology is used. For example, amammalian CCR2 protein or functional variant, an antibody or functionalportion thereof of the present invention, and a phage (e.g., a phage orcollection of phage such as a library) displaying a polypeptide, can becombined under conditions appropriate for binding of the antibody orportion thereof to the mammalian CCR2 protein or variant (e.g., in asuitable binding buffer). Phage which can compete with the antibody orportion thereof and bind to the mammalian CCR2 protein or variant can bedetected or selected using standard techniques or other suitablemethods. Bound phage can be separated from receptor using a suitableelution buffer. For example, a change in the ionic strength or pH canlead to a release of phage. Alternatively, the elution buffer cancomprise a release component or components designed to disrupt bindingof compounds (e.g., one or more compounds which can disrupt binding ofthe displayed peptide to the receptor, such as a ligand, inhibitor,and/or promoter which competitively inhibits binding). Optionally, theselection process can be repeated or another selection step can be usedto further enrich for phage which bind receptor. The displayedpolypeptide can be characterized (e.g., by sequencing phage DNA). Thepolypeptides identified can be produced and further tested for binding,and for inhibitor or promoter function. Analogs of such peptides can beproduced which will have increased stability or other desirableproperties.

In one embodiment, phage expressing and displaying fusion proteinscomprising a coat protein with an N-terminal peptide encoded by randomsequence nucleic acids can be produced. Suitable host cells expressing amammalian CCR2 protein or variant and an anti-CCR2 antibody orfunctional portion thereof, are combined with the phage, bound phage areselected, recovered and characterized. (See e.g., Doorbar, J. and G.Winter, J. Mol. Biol., 244: 361 (1994) discussing a phage displayprocedure used with a G protein-coupled receptor).

Other sources of potential ligands or other substances which bind to, orinhibitors and/or promoters of, mammalian CCR2 proteins include, but arenot limited to, variants of CCR2 ligands, including naturally occurring,synthetic or recombinant variants of MCP-1, MCP-2, MCP-3 and/or MCP-4,substances such as other chemoattractants or chemokines, variantsthereof, low molecular weight organic molecules, other inhibitors and/orpromoters (e.g., anti-CCR2 antibodies, antagonists, agonists), other Gprotein-coupled receptor ligands, inhibitors and/or promoters (e.g.,antagonists or agonists), and soluble portions of a mammalian CCR2receptor, such as a suitable receptor peptide or analog which caninhibit receptor function (see e.g., Murphy, R. B., WO 94/05695).

Models of Inflammation

In vivo models of inflammation are available which can be used to assessthe effects of antibodies and fragments of the invention in vivo astherapeutic agents. For example, leukocyte infiltration upon intradermalinjection of a chemokine and an antibody or fragment thereof reactivewith mammalian CCR2 into a suitable animal, such as rabbit, mouse, rat,guinea pig or rhesus macaque can be monitored (see e.g., Van Damme, J.et al., J. Exp. Med., 176: 59-65 (1992); Zachariae, C. O. C. et al., J.Exp. Med. 171: 2177-2182 (1990); Jose, P. J. et al., J. Exp. Med. 179:881-887 (1994)). In one embodiment, skin biopsies are assessedhistologically for infiltration of leukocytes (e.g., eosinophils,granulocytes). In another embodiment, labeled cells (e.g., stablytransfected cells expressing a mammalian CCR2, labeled with ¹¹¹In forexample) capable of chemotaxis and extravasation are administered to theanimal. For example, an antibody or fragment to be assessed can beadministered, either before, simultaneously with or after ligand oragonist is administered to the test animal. A decrease of the extent ofinfiltration in the presence of antibody as compared with the extent ofinfiltration in the absence of inhibitor is indicative of inhibition.

Diagnostic and Therapeutic Applications

The antibodies and fragments of the present invention are useful in avariety of applications, including research, diagnostic and therapeuticapplications. In one embodiment, the antibodies are labeled with asuitable label (e.g., fluorescent label, chemiluminescent label, isotopelabel, antigen or epitope label or enzyme label). For instance, they canbe used to isolate and/or purify receptor or portions thereof, and tostudy receptor structure (e.g., conformation) and function.

In addition, the various antibodies of the present invention can be usedto detect CCR2 or to measure the expression of receptor, for example, onT cells (e.g., CD8+ cells, CD45RO+ cells), monocytes and/or on cellstransfected with a receptor gene. Thus, they also have utility inapplications such as cell sorting (e.g., flow cytometry, fluorescenceactivated cell sorting), for diagnostic or research purposes.

The anti-CCR2 antibodies of the present invention have value indiagnostic applications. An anti-CCR2 antibody or fragment thereof canbe used to monitor expression of this receptor in HIV infectedindividuals, similar to the way anti-CD4 has been used as a diagnosticindicator of disease stage.

Typically, diagnostic assays entail detecting the formation of a complexresulting from the binding of an antibody or fragment thereof to CCR2.For diagnostic purposes, the antibodies or antigen-binding fragments canbe labeled or unlabeled. The antibodies or fragments can be directlylabeled. A variety of labels can be employed, including, but not limitedto, radionuclides, fluorescers, enzymes, enzyme substrates, enzymecofactors, enzyme inhibitors and ligands (e.g., biotin, haptens).Numerous appropriate immunoassays are known to the skilled artisan (see,for example, U.S. Pat. Nos. 3,817,827; 3,850,752; 3,901,654 and4,098,876). When unlabeled, the antibodies or fragments can be detectedusing suitable means, as in agglutination assays, for example. Unlabeledantibodies or fragments can also be used in combination with another(i.e., one or more) suitable reagent which can be used to detectantibody, such as a labeled antibody (e.g., a second antibody) reactivewith the first antibody (e.g., anti-idiotype antibodies or otherantibodies that are specific for the unlabeled immunoglobulin) or othersuitable reagent (e.g., labeled protein A).

In one embodiment, the antibodies or fragments of the present inventioncan be utilized in enzyme immunoassays, wherein the subject antibody orfragment, or second antibodies, are conjugated to an enzyme. When abiological sample comprising a mammalian CCR2 protein is combined withthe subject antibodies, binding occurs between the antibodies and CCR2protein. In one embodiment, a sample containing cells expressing amammalian CCR2 protein, such as human blood, is combined with thesubject antibodies, and binding occurs between the antibodies and cellsbearing a human CCR2 protein comprising an epitope recognized by theantibody. These bound cells can be separated from unbound reagents andthe presence of the antibody-enzyme conjugate specifically bound to thecells can be determined, for example, by contacting the sample with asubstrate of the enzyme which produces a color or other detectablechange when acted on by the enzyme. In another embodiment, the subjectantibodies can be unlabeled, and a second, labeled antibody can be addedwhich recognizes the subject antibody.

Kits for use in detecting the presence of a mammalian CCR2 protein in abiological sample can also be prepared. Such kits will include anantibody or functional fragment thereof which binds to a mammalianCC-chemokine receptor 2 or portion of said receptor, as well as one ormore ancillary reagents suitable for detecting the presence of a complexbetween the antibody or fragment and CCR2 or portion thereof. Theantibody compositions of the present invention can be provided inlyophilized form, either alone or in combination with additionalantibodies specific for other epitopes. The antibodies, which can belabeled or unlabeled, can be included in the kits with adjunctingredients (e.g., buffers, such as Tris, phosphate and carbonate,stabilizers, excipients, biocides and/or inert proteins, e.g., bovineserum albumin). For example, the antibodies can be provided as alyophilized mixture with the adjunct ingredients, or the adjunctingredients can be separately provided for combination by the user.Generally these adjunct materials will be present in less than about 5%weight based on the amount of active antibody, and usually will bepresent in a total amount of at least about 0.001% weight based onantibody concentration. Where a second antibody capable of binding tothe monoclonal antibody is employed, such antibody can be provided inthe kit, for instance in a separate vial or container. The secondantibody, if present, is typically labeled, and can be formulated in ananalogous manner with the antibody formulations described above.

Similarly, the present invention also relates to a method of detectingand/or quantitating expression of a mammalian CCR2 or a portion of thereceptor by a cell, in which a composition comprising a cell or fractionthereof (e.g., membrane fraction) is contacted with an antibody orfunctional fragment thereof (e.g., 1D9 and/or 8G2) which binds to amammalian CCR2 or portion of the receptor under conditions appropriatefor binding of the antibody or fragment thereto, and binding ismonitored. Detection of the antibody, indicative of the formation of acomplex between antibody and CCR2 or a portion thereof, indicates thepresence of the receptor. Binding of antibody to the cell can bedetermined as described above under the heading “Binding Assays”, forexample. The method can be used to detect expression of CCR2 on cellsfrom an individual (e.g., in a sample, such as a body fluid, such asblood, saliva or other suitable sample). The level of expression of CCR2on the surface of T cells or monocytes can also be determined, forinstance, by flow cytometry, and the level of expression (e.g., stainingintensity) can be correlated with disease susceptibility, progression orrisk.

Chemokine receptors function in the migration of leukocytes throughoutthe body, particularly to inflammatory sites. Inflammatory cellemigration from the vasculature is regulated by a three-step processinvolving interactions of leukocyte and endothelial cell adhesionproteins and cell specific chemoattractants and activating factors(Springer, T. A., Cell, 76:301-314 (1994); Butcher, E. C., Cell,67:1033-1036 (1991); Butcher, E. C. and Picker, L. J., Science (Wash.D.C.), 272:60-66 (1996)). These are: (a) a low affinity interactionbetween leukocyte selectins and endothelial cell carbohydrates; (b) ahigh-affinity interaction between leukocyte chemoattractant receptorsand chemoattractant/activating factors; and (c) a tight-binding betweenleukocyte integrins and endothelial cell adhesion proteins of theimmunoglobulin superfamily. Different leukocyte subsets expressdifferent repertoires of selectins, chemoattractant receptors andintegrins. Additionally, inflammation alters the expression ofendothelial adhesion proteins and the expression of chemoattractant andleukocyte activating factors. As a consequence, there is a great deal ofdiversity for regulating the selectivity of leukocyte recruitment toextravascular sites. The second step is crucial in that the activationof the leukocyte chemoattractant receptors is thought to cause thetransition from the selectinmediated cell rolling to theintegrin-mediated tight binding. This results in the leukocyte beingready to transmigrate to perivascular sites. Thechemoattractant/chemoattractant receptor interaction is also crucial fortransendothelial migration and localization within a tissue (Campbell,J. J., et al., J. Cell Biol., 134:255-266 (1996); Carr, M. W., et al.,Immunity, 4:179-187 (1996)). This migration is directed by aconcentration gradient of chemoattractant leading towards theinflammatory focus.

CCR2 has an important role in leukocyte trafficking. It is likely thatCCR2 is a key chemokine receptor for T cell or T cell subset or monocytemigration to certain inflammatory sites, and so anti-CCR2 mAbs can beused to inhibit (reduce or prevent) T cell or monocyte migration,particularly that associated with T cell dysfunction, such as autoimmunedisease, or allergic reactions or with monocyte-mediated disorders suchas atherosclerosis. Accordingly, the antibodies and fragments thereof ofthe present invention can also be used to modulate receptor function inresearch and therapeutic applications. For instance, the antibodies andfunctional fragments described herein can act as inhibitors to inhibit(reduce or prevent) (a) binding (e.g., of a ligand, an inhibitor or apromoter) to the receptor, (b) a receptor signaling function, and/or (c)a stimulatory function. Antibodies which act as inhibitors of receptorfunction can block ligand or promoter binding directly or indirectly(e.g., by causing a conformational change). For example, antibodies caninhibit receptor function by inhibiting binding of a ligand, or bydesensitization (with or without inhibition of binding of a ligand).Antibodies which bind receptor can also act as agonists of receptorfunction, triggering or stimulating a receptor function, such as asignaling and/or a stimulatory function of a receptor (e.g., leukocytetrafficking) upon binding to receptor.

Thus, the present invention provides a method of inhibiting leukocytetrafficking in a mammal (e.g., a human patient), comprisingadministering to the mammal an effective amount of an antibody orfunctional fragment of the present invention. Administration of anantibody or fragment of the present invention can result in ameliorationor elimination of the disease state.

The antibody of the present invention, or a functional fragment thereof,can also be used to treat disorders in which activation of the CCR2receptor by binding of chemokines is implicated. For example, theantibodies or functional fragments thereof (e.g., 1D9 and/or 8G2 orfunctional fragments thereof) can be used to treat allergy,atherogenesis, anaphylaxis, malignancy, chronic and acute inflammation,histamine and IgE-mediated allergic reactions, shock, and rheumatoidarthritis, atherosclerosis, multiple sclerosis, allograft rejection,fibrotic disease, asthma, and inflammatory glomerulopathies.

Diseases or conditions of humans or other species which can be treatedwith inhibitors of CCR2 receptor function (including antibodies orsuitable fragments thereof), include, but are not limited to:

inflammatory or allergic diseases and conditions, including respiratoryallergic diseases such as asthma, allergic rhinitis, hypersensitivitylung diseases, hypersensitivity pneumonitis, interstitial lung diseases(ILD) (e.g., idiopathic pulmonary fibrosis, or ILD associated withrheumatoid arthritis, systemic lupus erythematosus, ankylosingspondylitis, systemic sclerosis, Sjogren's syndrome, polymyositis ordermatomyositis); anaphylaxis or hypersensitivity responses, drugallergies (e.g., to penicillin, cephalosporins), insect sting allergies;inflammatory bowel diseases, such as Crohn's disease and ulcerativecolitis; spondyloarthropathies; scleroderma; psoriasis and inflammatorydermatoses such as dermatitis, eczema, atopic dermatitis, allergiccontact dermatitis, urticaria; vasculitis (e.g., necrotizing, cutaneous,and hypersensitivity vasculitis);

autoimmune diseases, such as arthritis (e.g., rheumatoid arthritis,psoriatic arthritis), multiple sclerosis, systemic lupus erythematosus,myasthenia gravis, juvenile onset diabetes, nephritides such asglomerulonephritis, autoimmune thyroiditis, Behcet's disease;

graft rejection (e.g., in transplantation), including allograftrejection or graft-versus-host disease, and organ transplant-associatedarteriosclerosis;

atherosclerosis;

cancers with leukocyte infiltration of the skin or organs;

stenosis or restenosis of the vasculature, particularly of the arteries,e.g., the coronary artery, such as stenosis or restenosis which resultsfrom vascular intervention (e.g., surgical, therapeutic or mechanicalintervention), as well as neointimal hyperplasia. For example,restenosis, which typically produces a narrowing of the lumenal openingof the vessel, can result from vascular injury including, but notlimited to, that produced by vascular graft procedures, angioplasty,including angioplasty performed by balloon, atherectomy, laser or othersuitable method (e.g., percutaneous translumenal coronary angioplasty(PTCA)), stent placement (e.g., mechanical or biological endovascularstent placement), vascular bypass procedures or combinations thereof, aswell as other procedures used to treat stenotic or occluded bloodvessels;

other diseases or conditions (including CCR2-mediated diseases orconditions), in which undesirable inflammatory responses are to beinhibited can be treated, including, but not limited to, reperfusioninjury, certain hematologic malignancies, cytokine-induced toxicity(e.g., septic shock, endotoxic shock), polymyositis, dermatomyositis,and granulomatous diseases including sarcoidosis.

Diseases or conditions of humans or other species which can be treatedwith promoters of CCR2 receptor function (including antibodies orfragments thereof), include, but are not limited to:

immunosuppression, such as that in individuals with immunodeficiencysyndromes such as AIDS, individuals undergoing radiation therapy,chemotherapy, therapy for autoimmune disease or other drug therapy(e.g., corticosteroid therapy), which causes immunosuppression; andimmunosuppression due congenital deficiency in receptor function orother causes.

Anti-CCR2 antibodies of the present invention can block the binding ofone or more chemokines, thereby blocking the downstream cascade of oneor more events leading to the above disorders.

Antibodies and functional fragments thereof which are antagonists ofCCR2 can be used as therapeutics for AIDS, as well as certaininflammatory diseases. HIV-1 and HIV-2 are the etiologic agents ofacquired immunodeficiency syndrome (AIDS) in humans. AIDS results inpart from the depletion of CD4+ T lymphocytes in HIV infectedindividuals. HIV-1 infects primarily T lymphocytes,monocytes/macrophages, dendritic cells and, in the central nervoussystem, microglia. All of these cells express the CD4 glycoprotein,which serves as a receptor for HIV-1 and HIV-2. Efficient entry of HIVinto target cells is dependent upon binding of the viral exteriorenvelope glycoprotein, gp120, to the amino-terminal CD4 domain. Aftervirus binding, the HIV-1 envelope glycoproteins mediate the fusion ofviral and host cell membranes to complete the entry process. Membranefusion directed by HIV-1 envelope glycoproteins expressed on theinfected cell surface leads to cell-cell fusion, resulting in syncytia.

Recently, host cell factors in addition to CD4 have been suggested todetermine the efficiency of HIV-1 envelope glycoprotein-mediatedmembrane fusion. The 7 transmembrane receptor (7TMR) termed HUMSTSR,LESTR, or “fusin” has been shown to allow a range of CD4-expressingcells to support infection and cell fusion mediated bylaboratory-adapted HIV-1 envelope glycoproteins (Feng, Y., et al.,Science (Wash. D.C.), 272:872-877 (1996)). Antibodies to HUMSTSR blockedcell fusion and infection by laboratory-adapted HIV-1 isolates but notby macrophagetropic primary viruses in vitro (Feng, Y., et al., Science(Wash. D.C.), 272:872-877 (1996)).

The ability of chemokine receptors and related molecules to facilitatethe infection of primary clinical HIV-1 isolates has been reportedrecently by several groups (see e.g., Bates, P., Cell, 86:1-3 (1996);Choe, H., et al., Cell, 85:1135-1148 (1996); Doranz et al., Cell85:1149-1158 (1996)). These studies indicated that involvement ofvarious members of the chemokine receptor family in the early stages ofHIV-1 infection helps to explain viral tropism and β-chemokineinhibition of primary HIV-1 isolates.

The present invention also provides a method of inhibiting HIV infectionof a cell (e.g., new infection and/or syncytium formation) whichexpresses a mammalian CCR2 or portion thereof, comprising contacting thecell with a composition comprising an effective amount of an antibody orfunctional fragment thereof which binds to a mammalian CCR2 or portionof said receptor. The composition can also comprise one or moreadditional agents effective against HIV, including, but not limited to,antiCCR3 antibodies, anti-CCR5 antibodies, and anti-fusin antibodies.

Various methods can be used to assess binding of HIV to a cell and/orinfection of a cell by HIV in the presence of the antibodies of thepresent invention. For example, assays which assess binding of gp120 ora portion thereof to the receptor, HIV infection and syncytium formationcan be used (see, for example, Choe, H., et al., Cell, 85:1135-1148(1996)). The ability of the antibody of the present invention to inhibitthese processes can be assessed using these or other suitable methods.

In addition, the present invention provides a method of treating HIV ina patient, comprising administering to the patient a compositioncomprising an effective amount of an antibody or functional fragmentthereof which binds to a mammalian CCR2 or portion of said receptor.Again, the composition can also comprise one or more additional agentseffective against HIV, including, but not limited to, anti-CCR3antibodies, anti-CCR5 antibodies, and anti-fusin antibodies. Therapeuticuse of antibody to treat HIV includes prophylactic use (e.g., fortreatment of a patient who may be or who may have been exposed to HIV).For example, health care providers who may be exposed or who have beenexposed to HIV (e.g., by needle-stick) can be treated according to themethod. Another example is the treatment of a patient exposed to virusafter unprotected sexual contact or failure of protection.

In AIDS, multiple drug treatment appears the most promising. Ananti-chemokine receptor antagonist that inhibits HIV infection can beadded to the drug treatment regimen, in particular by blocking virusinfection of new cells. Thus, administration of an antibody or fragmentof the present invention in combination with one or more othertherapeutic agents such as nucleoside analogues (e.g., AZT, 3TC, ddI)and/or protease inhibitors is envisioned, and provides an importantaddition to an HIV treatment regimen. In one embodiment, a humanizedanti-CCR2 mAb is used in combination with a (i.e., one or more)therapeutic agent to reduce viral load from patients, by preventingfusion and/or infection of new cells. Such an antibody can also beuseful in preventing perinatal infection.

Another aspect of the invention relates to a method of preventing HIVinfection in an individual, comprising administering to the individualan effective amount of an antibody or functional fragment thereof whichbinds to CCR2. According to the method, preventing HIV infectionincludes treatment in order to prevent (reduce or eliminate) infectionof new cells in an infected individual or in order to prevent infectionin an individual who may be, may have been, or has been, exposed to HIV.For example, individuals such as an HIV infected individual, a fetus ofan HIV infected female, or a health care worker may be treated accordingto the method of the present invention.

Modes of Administration

One or more antibodies or fragments of the present invention can beadministered to an individual by an appropriate route, either alone orin combination with (before, simultaneous with, or after) another drugor agent, or before, simultaneous with or after surgical, mechanical ortherapeutic intervention. For example, the antibodies of the presentinvention can also be used in combination with other monoclonal orpolyclonal antibodies (e.g., in combination with antibodies which bindother chemokine receptors, including, but not limited to, CCR3 and CCR5)or with existing blood plasma products, such as commercially availablegamma globulin and immune globulin products used in prophylactic ortherapeutic treatments. The antibodies or fragments of the presentinvention can be used as separately administered compositions given inconjunction with antibiotics and/or antimicrobial agents.

An effective amount of an antibody or fragment (i.e., one or moreantibodies or fragments) is administered. An effective amount is anamount sufficient to achieve the desired therapeutic (includingprophylactic) effect, under the conditions of administration, such as anamount sufficient for inhibition of a CCR2 function, and thereby,inhibition of an inflammatory response or HIV infection, or an amountsufficient for promotion of a CCR2 function, as indicated.

A variety of routes of administration are possible including, but notnecessarily limited to, oral, dietary, topical, parenteral (e.g.,intravenous, intraarterial, intramuscular, subcutaneous injection orinfusion), inhalation (e.g., intrabronchial, intraocular, intranasal ororal inhalation, intranasal drops), depending on the disease orcondition to be treated. Other suitable methods of administration canalso include rechargeable or biodegradable devices and slow releasepolymeric devices. The pharmaceutical compositions of this invention canalso be administered as part of a combinatorial therapy with otheragents.

Formulation of an antibody or fragment to be administered will varyaccording to the route of administration and formulation (e.g.,solution, emulsion, capsule) selected. An appropriate pharmaceuticalcomposition comprising an antibody or functional fragment thereof to beadministered can be prepared in a physiologically acceptable vehicle orcarrier. A mixture of antibodies and/or fragments can also be used. Forsolutions or emulsions, suitable carriers include, for example, aqueousor alcoholic/aqueous solutions, emulsions or suspensions, includingsaline and buffered media. Parenteral vehicles can include sodiumchloride solution, Ringer's dextrose, dextrose and sodium chloride,lactated Ringer's or fixed oils. A variety of appropriate aqueouscarriers are known to the skilled artisan, including water, bufferedwater, buffered saline, polyols (e.g., glycerol, propylene glycol,liquid polyethylene glycol), dextrose solution and glycine. Intravenousvehicles can include various additives, preservatives, or fluid,nutrient or electrolyte replenishers (See, generally, Remington'sPharmaceutical Science, 16th Edition, Mack, Ed. 1980). The compositionscan optionally contain pharmaceutically acceptable auxiliary substancesas required to approximate physiological conditions such as pH adjustingand buffering agents and toxicity adjusting agents, for example, sodiumacetate, sodium chloride, potassium chloride, calcium chloride andsodium lactate. The antibodies and fragments of this invention can belyophilized for storage and reconstituted in a suitable carrier prior touse according to art-known lyophilization and reconstitution techniques.The optimum concentration of the active ingredient(s) in the chosenmedium can be determined empirically, according to procedures well knownto the skilled artisan, and will depend on the ultimate pharmaceuticalformulation desired. For inhalation, the antibody or fragment can besolubilized and loaded into a suitable dispenser for administration(e.g., an atomizer, nebulizer or pressurized aerosol dispenser).

The present invention will now be illustrated by the following Examples,which are not intended to be limiting in any way. The teachings of allreferences cited herein are incorporated herein by reference.

EXAMPLES

Materials

The following materials were obtained from the indicated sources:

PE-conjugated anti-CD16, PE-conjugated streptavidin, and biotinylatedanti-human IgE were from Pharmingen (San Diego, Calif.). FITC-conjugatedgoat anti-mouse IgG was from Jackson Immunoresearch Laboratories (WestGrove, Pa.). FACS Lysing Buffer was from Becton Dickenson (MountainView, Calif.) and [¹²⁵I]-MCP-1 was from NEN (Boston, Mass.).

Cells, Cell Lines, and Tissue Culture

The murine pre-B lymphoma cell line L1/2 was maintained in RPMI-1640supplemented with 10% Fetal Clone I (Gibco BRL, Gaithersburg, Md.) 50Units/mL penicillin (Gibco BRL), 50 μg/mL streptomycin (Gibco BRL), 2 mML-Glutamine (Gibco BRL), and 55 μM β-mercaptoethanol (Gibco BRL). Othercell lines included transfectants of L1/2 cells expressing either CCR1(Campbell, J. et al. (1996) J. Cell Bio., 134:255-266), CCR5 (Wu et al.,Nature 384:179-183 (1996)) grown in the above culture mediumsupplemented with 800 μg/ml active G418. THP-1 cells (ATCC No. TIB202)were grown in accordance with ATCC instructions. PBMC were purified fromheparinized blood as described in Ponath et al., J. Clin. Invest.,97:604-612 (1996).

Preparation of CCR2 b Expression Construct and Stable Transfectants

The coding region for the human CCR2b (Charo et al. (1994) Proc. Natl.Acad. Sci. USA, 91:2752) was obtained by RT-PCR amplification asdescribed (Qin, S. et al. (1996) Eur. J. Immunol., 26:640-647). cDNA wasmade using oligo (dT)-priming, and amplification of the CCR2b codingregion was achieved by nested PCR with the following sets of primerswhich correspond to the positions of the CCR2b sequence (GenBankAccession No. U03905; Charo et al., Proc. Natl. Acad. Sci. USA91:2752-2756 (1994)) as indicated:

1) 5′ primer: 5′-TGAGACAAGCCACAAGCTGAAC-3′ (nucleotides 11 to 32; SEQ IDNO: 1);

3′ Primer: 5′-TCTGTATTAGTACACACAGCCC-3′ (nucleotides 1301 to 1280; SEQID NO: 2);

2) 5′ Primer: 5′-ATGCTGTCCACATCTCGTTCTCGG-3′ (nucleotides 81 to 104; SEQID NO: 3);

3′ Primer: 5′-TTATAAACCAGCCGAGACTTCCTGCTC-3′ (nucleotides 1164 to 1137;SEQ ID NO: 4).

The CCR2B cDNA coding region was modified to contain the CD5 signalpeptide leader sequence (Aruffo et al., Cell 61:1303-1313 (1990)). Thepredicted amino acid sequence of this peptide is:

NH₂-Met-Pro-Met-Gly-Ser-Leu-Gln-Pro-Leu-Ala-Thr-Leu-Tyr-Leu-Leu-Gly-Met-Leu-Val-Ala-Ser-Val-Leu-Ala. . . (SEQ ID NO: 5)

Using PCR with the CCR2b cDNA as template and two overlapping 5′ primersthat contain a BamHI restriction site, encode the CD5 signal peptidesequence and the amino terminal sequence of CCR2b, and a 3′ primerlocated internally in the CCR2b coding region.

5′ CD5 Seq1 primer

5′-GGGGATCCAGAAACCATGCCCATGGGGTCTCTGCAACCGCTGGCCACCT TGTACCTGCTG-3′ (SEQID NO: 6)

5′ CD5 Seq2 primer

5′-GCCACCTTGTACCTGCTGGGGATGCTGGTCGCTTCCGTGCTAGCGATGC TGTCCACATCTCGTTC-3′(SEQ ID NO: 7)

3′ CCR2AB2 primer-5′-GACGACCAGCATGTTGCC-3′ (SEQ ID NO: 8; U03905nucleotides 272 to 255)

The 278 base pair amplified fragment was digested with BamHI and Apaland the resulting 209 base pair fragment was inserted at the Apal siteat position 206 of the CCR2b cDNA (GenBank Accession No. U03905) toreplace the endogenous 5′ base pair fragment of CCR2. The resultingsequence that encodes a CCR2b with the CD5 signal peptide leadersequence immediately preceding the receptor initiator methionine wasinserted into the BamHI and Xhol sites of pcDNA3 (Invitrogen, San Diego,Calif.) to create the mammalian expression plasmid pCD5 MCPRB. TheCD5-CCR2b fragment was subcloned into the BamH I-Not I site of pCDEF3(Goldman et al., (1996) Biotechniques 21:1013-1015), and this constructwas designated CCR2bDEF3. In this expression vector, the expression ofthe inserted gene is driven by the EF-1α promoter.

Fifty milliliters of L1/2 cells were seeded at 4×10⁵ cells/mL the daybefore the electroporation. On the day of the electroporation, thecells, which had grown up to a density of 1×10⁶/mL, were centrifuged outof their medium and resuspended in 800 μl room temperatureelectroporation buffer (Zajac et al., DNA 7:509-513). 120 mM L-GlutamicAcid (Sigma), 7 mM Mg Acetate (EM Science), 4.3 mM Glucose (Sigma), 17mM K Pipes, pH 6.9 (Sigma), 1 mM EGTA (Sigma), 5 mM ATP, pH 7.0 (Sigma).Twenty-five micrograms Sca I linearized, phenol/chloroform/isoamylalcohol extracted and isopropanol precipitated CCR2bDEF3 plasmid DNA wasplaced in an 0.4 cm gap electroporation curvette. The resuspended cellswere added to the curvette, and a single pulse applied at 450 volts, 960μFd. The cells were then transferred from the curvette to a T-75 flaskcontaining 15 mL L1/2 growth medium (described above, and grown forthree days, at which time the cells were centrifuged out of their mediumand resuspended in L1/2 growth medium additionally supplemented with 1mM sodium pyruvate (Gibco BRL) and 0.8 mg/mL active G418 (Gibco BRL).

Selection of Cells Expressing CCR2b by Chemotaxis

The transfected cells were allowed to grow for eleven days, at whichpoint they were split 1:20 into fresh growth medium. On the sixteenthday, the cells were selected by chemotaxis. 600 μL 1 nM MCP-1 in RPMI1640 supplemented with 0.5% BSA (RPMI/BSA) was placed in the lowerchamber and 1×10⁶ CCR2bDEF3 cells in 100 μl of RPMI/BSA were placed inthe upper chamber of a 3.0 micron pore 24-well chemotaxis plate (BectonDickinson). The cells were allowed to chemotax for four hours and twentyminutes in a 37° C., 5% CO₂, humidified incubator, at which time theupper chamber was removed. This incubation time was chosen at the timeof the experiment because it was sufficiently long for cells respondingto the MCP-1 to chemotax, but short enough to keep the background low.

Secondary Selection of CCR2b Expressing-Cells by FACS Sorting

The cells which had chemotaxed through the membrane and into the lowerchamber were grown up, and further purified by sterile FACS sorting. Tenmillion CCR2bDEF3 cells were centrifuged out of their medium,resuspended in 2.5 mL PBS(+Ca, Mg) supplemented with 1% heat-inactivatedFetal Calf Serum (“HI FCS”) (Gibco BRL) and 2.5 mL sterile filteredanti-CCR2b amino-terminal peptide antibody supernatant 5A11. The cellsand the antibody were mixed and allowed to incubate on ice for thirtyminutes. The cells were then washed twice with PBS (+) (Gibco BRL), andresuspended in 5 mL of a sterile filtered, 1:250 dilution ofFITC-conjugated, affinity-purified F(ab¹)₂ goat anti-mouse lgG (JacksonImmunoResearch Laboratories) in PBS (+) supplemented with 1% HI FCS. Thecells were incubated for thirty minutes on ice in the dark, and thenwashed twice with PBS(+) (GIBCO BRL). The cells were sorted on theFACSCalibur® and the brightest 4% of cells were collected. (FL1≧3×10²).

The sorted cells were allowed to grow up, and they were resorted usingthe same protocol as above. The brightest 1% of cells were collected.(FL1≧3×10³).

Monoclonal Antibody Production

To produce mAbs to CCR2b, transfectants were continually monitored toensure that levels of expression did not drift downward. FACS stainingwas performed periodically to ascertain receptor expression on thetransfectants using the anti CCR2b antibody supernatant 5A11 with goatanti-mouse IgG FITC as the secondary antibody.

Twenty million CCR2bDEF3.L1/2 cells were washed in RPMI 1640 (Gibco BRL)and incubated in RPMI 1640 plus 0.2 mg/mL Mitomycin C for 30 minutes at37° C. The cells were then washed twice with PBS (+) and 2×10⁷ cells in0.5 mL PBS (+) were injected intraperitoneally into a C57 BL/6 femalemouse. This was repeated two more times at two week intervals. Thefourth time, 2×10⁷ cells were resuspended in 0.25 mL and injectedintravenously. Three days after the intravenous injection, the mouse wassacrificed and the spleen removed and the cells fused with the SP2/0cell line as described (Current Protocols in Immunology, John Wiley andSons, New York, 1992).

This set of mice had previously been immunized many times with 2different cell lines as well as a synthetic peptide, but no antibodiesthat stained CCR2 positive cells were generated from several fusions.The above four immunizations with the CCR2bDEF3.L1/2 cell lineexpressing high levels of CCR2b were critical to obtain the describedantibody.

Selecting Single Cell Clone of CCR2 Transfectants by Limiting Dilution

After the mouse received the last injection, the twice sorted cells wereallowed to grow up again, and then they were further purified bylimiting dilution. The cells were plated at 1 and 0.5 cell per well in96 well plates. Subcloned cells from the 0.5 cell per well dilution weregrown up and tested for CCR2b expression by indirect immunofluorescentFACS analysis using the anti-CCR2b antibody supernatant 5A11 with goatanti-mouse IgG FITC as the secondary antibody. The procedure was thesame as described above, except that the staining volume was 100 μl.Four positives were selected and frozen down.

Identification of Positive Monoclonal Antibodies

Immunofluorescent staining analysis using a FACScan® (Becton Dickinson &Co., Mountain View, Calif.) was used to identify the monoclonalantibodies which were reactive with the CCR2b receptor. Hybridomaculture supernatants were assayed in a 96-well format using goatanti-mouse IgG FITC as the secondary antibody. CCR2bDEF3.L1/2 cells wereused to identify monoclonal antibodies reactive with CCR2b, anduntransfected L1/2 cells were used to eliminate monoclonal antibodiesreactive with other cell surface proteins.

FACS Staining—Cultured Cells

For the staining of cultured transfectant cell lines 0.5×10⁶ cells in 50μl were resuspended in PBS+1% FCS in a 96 well polystyrene V-bottomplate. 50 μl of primary antibody supernatants or HT medium (negativecontrol) were added, and the samples were incubated at 4° C. for 30 min.100 μl of PBS were added and the cells were pelleted by centrifugationand washed once with PBS. The pellet was resuspended in 100 μl PBS+1%FCS containing FITC-conjugated goat anti-mouse IgG antibody (a 1:250dilution) and incubated for thirty minutes at 4° C. in the dark. Thecells were washed twice with PBS, resuspended in PBS, and analyzed byflow cytrometry with a FacScan cytometer using the CellQuest software(Becton-Dickenson) Cells were fixed with PBS/1% formaldehyde if theywere not to be analyzed the same day. Monoclonal antibodies 1D9 and 8G2stain CCR2 transfectants but not CCR1 or CCR5 transfectants (FIGS.1A-1O).

FACS Staining—Whole Blood

100 μl whole blood was mixed with 100 μL of 1D9 antibody hybridomasupernatants or HT medium (negative control) and incubated at 4° C. for30 min. After one wash with PBS, 100 μL FITC-conjugated goat anti-mouseIgG antibody (a 1:250 dilution) was added to each sample and incubatedfor 30 min. at 4° C. in the dark. Samples were then washed once with PBSif a second color staining is to be done, otherwise washed twice more inPBS. For two color staining 5 μl of mouse serum was added to the cellpellets after the single wash, mixed, and incubated for five minutes at4° C. in the dark. Second primary antibodies (or PBS as a negativecontrol) were added (10 μl anti-CD16, 100 μl 1:200 dilution of anti-IgE)and incubated for thirty minutes at 4° C. in the dark. Samples were thenwashed one time with PBS and resuspended in 100 μL streptavidin PE(1:200 PBS+1% BSA) and incubated for fifteen minutes at 4° C. in thedark. Eyrythrocytes were lysed by adding 2 ml of FACS Lysing Buffer toeach sample and incubating at room temperature in the dark for fifteenminutes or until samples were clear. The cells were pelleted bycentrifugation and all but 200 μl of the supernatant was aspirated. Thesamples were analyzed by flow cytometry on a FacScan cytometer using theCellQuest software (Becton-Dickenson). CCR2b is expressed on mostmonocytes, a subpopulation of lymphocytes and a subset of granulocytes(FIGS. 2A-2L). CCR2b is expressed on an IgE-positive population inperipheral blood (basophils) (FIGS. 3A-3I).

MCP-1 Binding Assays

MCP-1 binding was performed in a final volume of 0.1 ml of 50 mM HepespH 7.4, 1 mM CaCl₂, 5 mM MgCl₂, 0.02% sodium azide, 0.5% BSA (HBB),containing either 2.5 μg THP-1 membrane protein or 500,000 PBMC and 0.1nM of [¹²⁵]-MCP-1. Competition binding experiments were performed byincluding variable concentrations of unlabeled MCP-1, 1D9 antibody, or anegative control IgG2a. Nonspecific binding was determined following theaddition of a 2500-fold excess of unlabeled MCP-1. Samples wereincubated for 60 minutes at room temperature, and bound and free tracerwere separated by filtration through 96-well GF/B filterplates presoakedin 0.3% polyethyleneimine. The filters were washed in HBB furthersupplemented with 0.5 M NaCl, dried, and the amount of boundradioactivity determined by liquid scintillation counting. mAb 1D9inhibits [¹²⁵I]MCP-1 binding to THP-1 cell membranes with an IC₅₀ ofabout 0.004 μg/ml (approximately 0.02 nM; FIG. 4) and to fresh PBMC withan IC₅₀ of 0.04 μg/ml (approximately 0.2 nM; FIG. 5).

Chemotaxis of PBMC

Chemotaxis was assayed using a 3 μm pore size 96-well chemotaxis plate(Neuroprobe, Cabin John, Md.). PBMC isolated by standard methods usingFicoll-Hypaque density gradient centrifugation were washed withPBS/0.5%BSA and then resuspended in chemotaxis assay media (HBSS/10 mMHEPES/0.5% Fatty acid free BSA) to a final concentration of 10×10⁶cells/ml. Cells were princubated in chemotaxis assay media at roomtemperature for 20 min. with various concentrations of the anti-CCR2antibody, 1D9, or nonspecific murine IgG2a. The same dilutions ofantibody were mixed with chemokine and 30 μl of the mixture was added toeach of the bottom wells of the chemotaxis plate. The bottom wells arecovered with the membrane, and 25 μl of the cell and antibody mixtureare added to the top of the filter. The plates are incubated at 37° C.in 5% CO₂ incubator for approximately 80 min. At the completion of themigration, the membrane is removed and the plate with the bottom wellsis incubated −80 C. for 30 minutes to freeze the contents. The platesare thawed at 37° C. for 10 minutes. 6 μl of a 1:400 dilution of CyQuantreagent (Molecular Probes, Eugene, Oreg.) in a lysis buffer provided bythe supplier is added to each well, and the cell migration is quantifiedas indicated by fluorescence intensity determined using a CytoFlourfluorescence plate reader at 485ex/535em. mAb 1D9 inhibits MCP-1-inducedchemotaxis, but not RANTES-induced chemotaxis, of fresh PBMC (FIGS. 6Aand 6B). Inhibition of MCP-1-induced chemotaxis of fresh PBMC has beendemonstrated with 10 μg/ml (≈40 nM).

While this invention has been particularly shown and described withreference to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the spirit and scope of theinvention as defined by the appended claims.

8 1 22 DNA Artificial Sequence Primer 1 gagacaagc cacaagctga ac 22 2 22DNA Artificial Sequence Primer 2 tctgtattag tacacacagc cc 22 3 24 DNAArtificial Sequence Primer 3 atgctgtcca catctcgttc tcgg 24 4 27 DNAArtificial Sequence Primer 4 ttataaacca gccgagactt cctgctc 27 5 24 PRTHomo sapiens 5 Met Pro Met Gly Ser Leu Gln Pro Leu Ala Thr Leu Tyr LeuLeu Gly 1 5 10 15 Met Leu Val Ala Ser Val Leu Ala 20 6 60 DNA ArtificialSequence Primer 6 ggggatccag aaaccatgcc catggggtct ctgcaaccgc tggccaccttgtacctgctg 60 7 65 DNA Artificial Sequence Primer 7 gccaccttgtacctgctggg gatgctggtc gcttccgtgc tagcgatgct gtccacatct 60 cgttc 65 8 18DNA Artificial Sequence Primer 8 gacgaccagc atgttgcc 18

What is claimed is:
 1. A method of inhibiting restenosis in a patient,comprising administering to the patient an effective amount of anantibody or antigen-binding fragment thereof which binds to mammalianCC-chemokine receptor 2, wherein said antibody or antigen-bindingfragment thereof inhibits binding of a ligand to the receptor.
 2. Amethod according to claim 1, wherein the antibody or antigen-bindingfragment thereof is selected from the group consisting of: a) monoclonalantibody 1D9; b) an antibody having the epitopic specificity ofmonoclonal antibody 1D9; c) an antibody which can compete withmonoclonal antibody 1D9 for binding to mammalian CC-chemokine receptor2; d) monoclonal antibody 8G2; e) an antibody having the epitopicspecificity of monoclonal antibody 8G2; f) an antibody which can competewith monoclonal antibody 8G2 for binding to mammalian CC-chemokinereceptor 2; g) antigen-binding fragments of any one of (a) through (g)which bind to mammalian CC-chemokine receptor 2; and h) combinations ofthe foregoing.
 3. A method of inhibiting restenosis of a vessel in amammal, comprising administering to said mammal an effective amount ofan antibody or antigen-binding fragment thereof which binds to amammalian CC-chemokine receptor 2, wherein said antibody orantigen-binding fragment thereof inhibits binding of a ligand to thereceptor.
 4. A method according to claim 3 wherein the antibody orantigen-binding fragment thereof is selected from the group consistingof: a) monoclonal antibody 1D9; b) an antibody having the epitopicspecificity of monoclonal antibody 1D9; c) an antibody which can competewith monoclonal antibody 1D9 for binding to mammalian CC-chemokinereceptor 2; d) monoclonal antibody 8G2; e) an antibody having theepitopic specificity of monoclonal antibody 8G2; f) an antibody whichcan compete with monoclonal antibody 8G2 for binding to mammalianCC-chemokine receptor 2; g) antigen-binding fragments of any one of (a)through (g) which bind to mammalian CC-chemokine receptor 2; and h)combinations of the foregoing.
 5. A method according to claim 3, whereinsaid restenosis is associated with vascular intervention in said mammal.6. A method according to claim 5, wherein said vascular interventioncomprises angioplasty.
 7. A method according to claim 5, wherein saidvascular intervention comprises stent placement.
 8. A method accordingto claim 5, wherein said vascular intervention comprises angioplasty andstent placement.
 9. A method of inhibiting narrowing of the lumen of avessel in a mammal, comprising administering to said mammal an effectiveamount of an antibody or antigen-binding fragment thereof which binds toa mammalian CC-chemokine receptor 2, wherein said antibody orantigen-binding fragment thereof inhibits binding of a ligand to thereceptor.
 10. A method according to claim 9 wherein the antibody orantigen-binding fragment thereof is selected from the group consistingof: a) monoclonal antibody 1D9; b) an antibody having the epitopicspecificity of monoclonal antibody 1D9; c) an antibody which can competewith monoclonal antibody 1 D9 for binding to mammalian CC-chemokinereceptor 2; d) monoclonal antibody 8G2; e) an antibody having theepitopic specificity of monoclonal antibody 8G2; f) an antibody whichcan compete with monoclonal antibody 8G2 for binding to mammalianCC-chemokine receptor 2; g) antigen-binding fragments of any one of (a)through (g) which bind to mammalian CC-chemokine receptor 2; and h)combinations of the foregoing.
 11. A method of inhibiting neointimalhyperplasia of a vessel in a mammal, comprising administering to saidmammal an effective amount of an antibody or antigen-binding fragmentthereof which binds to a mammalian CC-chemokine receptor 2, wherein saidantibody or antigen-binding fragment thereof inhibits binding of aligand to the receptor.
 12. A method according to claim 11 wherein theantibody or antigen-binding fragment thereof is selected from the groupconsisting of: a) monoclonal antibody 1D9; b) an antibody having theepitopic specificity of monoclonal antibody 1D9; c) an antibody whichcan compete with monoclonal antibody 1D9 for binding to mammalianCC-chemokine receptor 2; d) monoclonal antibody 8G2; e) an antibodyhaving the epitopic specificity of monoclonal antibody 8G2; f) anantibody which can compete with monoclonal antibody 8G2 for binding tomammalian CC-chemokine receptor 2; g) antigen-binding fragments of anyone of (a) through (g) which bind to mammalian CC-chemokine receptor 2;and h) combinations of the foregoing.
 13. A method according to claim11, wherein said neointimal hyperplasia is associated with vascularintervention in said mammal.
 14. A method according to claim 13, whereinsaid vascular intervention comprises angioplasty.
 15. A method accordingto claim 13, wherein said vascular intervention comprises stentplacement.
 16. A method according to claim 13, wherein said vascularintervention comprises angioplasty and stent placement.
 17. A methodaccording to claim 1, wherein the ligand is a chemokine.
 18. A methodaccording to claim 17, wherein the chemokine is selected from the groupconsisting of MCP-1, MCP-2, MCP-3, MCP-4 and combinations thereof.
 19. Amethod according to claim 1, wherein said antibody or antigen-bindingfragment thereof binds the amino-terminal domain of the receptor.
 20. Amethod according to claim 19, wherein said antibody or antigen-bindingfragment binds a portion of the amino-terminal domain which is fromabout amino acid 1 to about amino acid 30 of the receptor.
 21. A methodaccording to claim 1, wherein said mammalian CC-chemokine receptor 2 isa human CC-chemokine receptor
 2. 22. A method according to claim 1,wherein said antibody or antigen-binding fragment thereof is amonoclonal antibody or antigen-binding fragment thereof.
 23. A methodaccording to claim 1, wherein said antibody or antigen-binding fragmentthereof is a human antibody or antigen-binding fragment thereof.
 24. Amethod according to claim 1, wherein said antibody or antigen-bindingfragment is a chimeric antibody or antigen-binding fragment thereof. 25.A method according to claim 1, wherein said antibody or antigen-bindingfragment is a recombinant antibody or antigen-binding fragment thereof.26. A method according to claim 1, wherein said antibody orantigen-binding fragment is a humanized antibody or antigen-bindingfragment thereof.
 27. A method according to claim 26, wherein saidhumanized antibody comprises one or more antigen-binding regions ofmonoclonal antibody 1D9.
 28. A method according to claim 26, whereinsaid humanized antibody comprises one or morecomplementarity-determining regions of monoclonal antibody 1D9.
 29. Amethod according to claim 28, wherein said humanized antibody comprisessix complementarity-determining regions of monoclonal antibody 1D9. 30.A method according to claim 1, wherein said antigen-binding fragment isselected from the group consisting of an Fv fragment, an Fab fragment,an Fab′ fragment and an F(ab′)₂ fragment.
 31. A method according toclaim 3, wherein the ligand is a chemokine.
 32. A method according toclaim 31, wherein the chemokine is selected from the group consisting ofMCP-1, MCP-2, MCP-3, MCP-4 and combinations thereof.
 33. A methodaccording to claim 3, wherein said antibody or antigen-binding fragmentthereof binds the amino-terminal domain of the receptor.
 34. A methodaccording to claim 33, wherein said antibody or antigen-binding fragmentbinds a portion of the amino-terminal domain which is from about aminoacid 1 to about amino acid 30 of the receptor.
 35. A method according toclaim 3, wherein said mammalian CC-chemokine receptor 2 is a humanCC-chemokine receptor
 2. 36. A method according to claim 3, wherein saidantibody or antigen-binding fragment thereof is a monoclonal antibody orantigen-binding fragment thereof.
 37. A method according to claim 3,wherein said antibody or antigen-binding fragment thereof is a humanantibody or antigen-binding fragment thereof.
 38. A method according toclaim 3, wherein said antibody or antigen-binding fragment is a chimericantibody or antigen-binding fragment thereof.
 39. A method according toclaim 3, wherein said antibody or antigen-binding fragment is arecombinant antibody or antigen-binding fragment thereof.
 40. A methodaccording to claim 3, wherein said antibody or antigen-binding fragmentis a humanized antibody or antigen-binding fragment thereof.
 41. Amethod according to claim 40, wherein said humanized antibody comprisesone or more antigen-binding regions of monoclonal antibody 1D9.
 42. Amethod according to claim 40, wherein said humanized antibody comprisesone or more complementarity-determining regions of monoclonal antibody1D9.
 43. A method according to claim 42, wherein said humanized antibodycomprises six complementarity-determining regions of monoclonal antibody1D9.
 44. A method according to claim 3, wherein said antigen-bindingfragment is selected from the group consisting of an Fv fragment, an Fabfragment, an Fab′ fragment and an F(ab′)₂ fragment.
 45. A methodaccording to claim 9, wherein the ligand is a chemokine.
 46. A methodaccording to claim 45, wherein the chemokine is selected from the groupconsisting of MCP-1, MCP-2, MCP-3, MCP-4 and combinations thereof.
 47. Amethod according to claim 9, wherein said antibody or antigen-bindingfragment thereof binds the amino-terminal domain of the receptor.
 48. Amethod according to claim 47, wherein said antibody or antigen-bindingfragment binds a portion of the amino-terminal domain which is fromabout amino acid 1 to about amino acid 30 of the receptor.
 49. A methodaccording to claim 9, wherein said mammalian CC-chemokine receptor 2 isa human CC-chemokine receptor
 2. 50. A method according to claim 9,wherein said antibody or antigen-binding fragment thereof is amonoclonal antibody or antigen-binding fragment thereof.
 51. A methodaccording to claim 9, wherein said antibody or antigen-binding fragmentthereof is a human antibody or antigen-binding fragment thereof.
 52. Amethod according to claim 9, wherein said antibody or antigen-bindingfragment is a chimeric antibody or antigen-binding fragment thereof. 53.A method according to claim 9, wherein said antibody or antigen-bindingfragment is a recombinant antibody or antigen-binding fragment thereof.54. A method according to claim 9, wherein said antibody orantigen-binding fragment is a humanized antibody or antigen-bindingfragment thereof.
 55. A method according to claim 54, wherein saidhumanized antibody comprises one or more antigen-binding regions ofmonoclonal antibody 1D9.
 56. A method according to claim 54, whereinsaid humanized antibody comprises one or morecomplementarity-determining regions of monoclonal antibody 1D9.
 57. Amethod according to claim 56, wherein said humanized antibody comprisessix complementarity-determining regions of monoclonal antibody 1D9. 58.A method according to claim 9, wherein said antigen-binding fragment isselected from the group consisting of an Fv fragment, an Fab fragment,an Fab′ fragment and an F(ab′)₂ fragment.
 59. A method according toclaim 11, wherein the ligand is a chemokine.
 60. A method according toclaim 59, wherein the chemokine is selected from the group consisting ofMCP-1, MCP-2, MCP-3, MCP-4 and combinations thereof.
 61. A methodaccording to claim 11, wherein said antibody or antigen-binding fragmentthereof binds the amino-terminal domain of the receptor.
 62. A methodaccording to claim 61, wherein said antibody or antigen-binding fragmentbinds a portion of the amino-terminal domain which is from about aminoacid 1 to about amino acid 30 of the receptor.
 63. A method according toclaim 11, wherein said mammalian CC-chemokine receptor 2 is a humanCC-chemokine receptor
 2. 64. A method according to claim 11, whereinsaid antibody or antigen-binding fragment thereof is a monoclonalantibody or antigen-binding fragment thereof.
 65. A method according toclaim 11, wherein said antibody or antigen-binding fragment thereof is ahuman antibody or antigen-binding fragment thereof.
 66. A methodaccording to claim 11, wherein said antibody or antigen-binding fragmentis a chimeric antibody or antigen-binding fragment thereof.
 67. A methodaccording to claim 11, wherein said antibody or antigen-binding fragmentis a recombinant antibody or antigen-binding fragment thereof.
 68. Amethod according to claim 11, wherein said antibody or antigen-bindingfragment is a humanized antibody or antigen-binding fragment thereof.69. A method according to claim 68, wherein said humanized antibodycomprises one or more antigen-binding regions of monoclonal antibody1D9.
 70. A method according to claim 68, wherein said humanized antibodycomprises one or more complementarity-determining regions of monoclonalantibody 1D9.
 71. A method according to claim 70, wherein said humanizedantibody comprises six complementarity-determining regions of monoclonalantibody 1D9.
 72. A method according to claim 11, wherein saidantigen-binding fragment is selected from the group consisting of an Fvfragment, an Fab fragment, an Fab′ fragment and an F(ab′)₂ fragment.